EA010258B1 - Drug discovery method - Google Patents

Abstract

A method of obtaining information about a chemically active area of a target molecule, for example for drug discovery, comprising: providing a set of substantially rigid chemical gauges; reacting said target with a plurality of gauges of said set of gauges; assaying a binding of said gauges with said target to obtain a plurality of assay results; and analyzing said assay results to obtain information about said chemically active area.

Description

FIELD OF THE INVENTION

The present invention relates to methods for determining the affinity of molecules, for example, for use in the search for new drugs.

State of the art

Developing a new pharmaceutical product, from concept to ready for sale, usually costs hundreds of millions of dollars and takes many years. The development process begins with the step of selecting a molecule (potential pharmaceutical preparation) for the target, i.e. protein of the human body or microorganism. Matching a molecule to a pharmaceutical is known as determining the leader of a drug because it can lead to drug development. The molecule is then modified for greater activity, greater selectivity and greater pharmaceutical suitability (for example, less toxicity and an easier route of administration). The failure rate at these stages is very high.

With the development of combinatorial chemistry and automated screening techniques, a new method for the search for a drug substance has been developed. In such a new method, a large library of molecules is chemically tested against a target with the molecule having the best match, used as a starting point for finding a leader and / or as a leader. Some of these libraries are empirically constructed, for example, based on available molecules and / or molecules whose action is known as pharmaceuticals. Other libraries are designed to have as wide a range of molecules as possible. Other libraries are designed so that individual molecules can have the greatest chance of matching the target as much as possible. Typically, molecules are selected in such a way that they are as different as possible and similar to a drug substance (for example, size, chemical behavior) so that when a match is found, they can serve as a leader.

Some links to such libraries and / or other search methods include P1ske !! 8.Ό. e! A1., 1. Siet. Ιπί. Erase !. 8s1. 36 (6), p. 1214-23 (1996) and Regdikop A.M. e! A1., 1. Vyuto1. 8g 1 (2), p. 65 (1996), Vichy A.V. e! A1., Proc. No. 111. Asab. 8s1. I8A 91, p. 4708-12 (1994), E11stap 1. e! A1., Proc. No. 111. Asab. Ac1. I8A 94, p.2779-82 (1997) and Ma1u Ό.Ε e! A1., Proc. No. 111. Asab. 8sk I8A 97 (6), p. 2419-24 (2000), the descriptions of which are incorporated into this description by reference.

Another virtual type of screening based on structure is known. In the virtual method, a target model is created (for example, using x-ray crystallography, evaluation of the tertiary structure, analogy). The affinity of a large number of molecules is then determined by calculating the behavior when the model of the molecule is docked with the target model. Due to the relatively primitive state of molecular modeling and the lack of available models, this method is currently not particularly successful.

8ipe515. 1ps., In Ό.Ε Ma1u e! a1. RYA8 97 (6), p. 2419-24 (2000), the description of which is incorporated into this description by reference, proposes to use large fragments of molecules as leaders, followed by joining together such found corresponding leaders into larger leaders, which are again tested for compliance . Fragments are provided with predetermined linkers for joining together.

PCT Application PCT / I899 / 06734 (UO 99/49314), the disclosure of which is incorporated herein by reference, also describes a pattern for using fragments followed by combining fragments to provide leaders.

Disclosure of invention

An aspect of some embodiments relates to a method for characterizing targets in which a plurality of small measurement molecules interact with a target and the target is characterized based on an analysis of the interactions of the measurement molecules with the target. In an illustrative embodiment of the present invention, none of the measuring molecules is used as a leader or as a fragment of a leader, and is not a molecule selected for interaction based on their diversity in the type of drug substance. Rather, the measuring molecules are selected based on the expected ability to measure various chemical and / or physical sizes of the target. In an illustrative embodiment of the present invention, since the number of measuring molecules is relatively small (eg, <10 ⁶ ), such a quantity encompasses the characterization space of the target molecule and may be sufficient to provide relatively complete determination of the characteristics of the target. In other embodiments, only a partial characterization is required and / or achieved. Alternatively or additionally, since the measuring molecules are selected for reasons of breadth, they are also used as leaders or as fragments of a leader.

In an illustrative embodiment of the present invention, a complete method for finding a drug substance comprises:

(a) target selection;

(i) optionally selecting a set of measuring molecules used for the target, or using

- 1 010258 universal library;

(c) characterization of the target using a set of measuring molecules;

(b) reconstruction of the pharmaceutical model of the target based on characterization; and (e) using the model in a search method, for example, to select, reject, filter, and / or develop a drug leader.

In some embodiments of the present invention, conventional measurement molecules can perform one or more measurements, and a processing method, such as clustering, is not necessarily used to extract specific measurements made with the molecules.

In an illustrative embodiment of the present invention, the measuring molecules are a set of chemical meters, of which some, usually a small amount, bind to the target, usually to one or more active sites of the target. The binding of the meter to the target can be determined using various analysis methods, including essentially any method known in the art, for example, by searching for a change in the chemical or biological behavior of the target or by searching for a decrease in the number of free measuring molecules in the sample. In a specific example, a functional analysis of a protease (e.g., Ηΐν protein) comprises attaching a fluorescent molecule to a protein (or other peptide). The protease provides the opportunity to interact with the meter, and it is suggested that such an interaction will reduce or neutralize (or enhance) its affinity for the protein, and the change in affinity can be determined by measuring fluorescent proteases (e.g. polarization) in a mixture of protein and protease. In an illustrative embodiment of the present invention, each meter is selected so that it has an affinity for one or more specific geometric arrangements. In an illustrative embodiment of the present invention, the overall geometry of the target region is reconstructed from determining the affinity (and / or lack of affinity) of a plurality of meters.

In an illustrative embodiment of the present invention, each of the meters is constructed from a frame to which many specific chemical groups are attached. Three such groups define a triangle of groups, which includes both the definition of groups at the vertices and the determination of the distance between the vertices. In an illustrative embodiment of the present invention, the frames and groups are selected so that the triangles are relatively rigid, however, some degree of nonrigidity of the lengths of the sides of the triangle (distance between groups) may be desirable.

Each such triangle of groups corresponds to a specific spatial arrangement of the three binding sites that correspond to the groups. Optionally, the distance between the groups is changed for different meters so as to provide a range of triangles with different desired combinations of groups and the distances between the groups. As will be shown below, the gauge library, which includes a comprehensive set of such triangles for measurements, with respect to both distance and groups, is not excessively large.

In an exemplary embodiment of the present invention, the framework and / or groups are selected so as to have minimal flexibility so that they more specifically determine the geometric characteristics to which they correspond.

Scaffolds and / or groups are optionally selected so that they have a low molecular weight in order to improve the connection of meters and / or targets with low affinity and possibly provide information for such cases.

In an illustrative embodiment of the present invention, when selecting meters for a measurement library, some degree of overlap of the triangle with the groups is provided. For example, an overlap repetition factor of 2 or 3 can be provided (for example, each triangle is present in at least 2 or 3 meters). It is believed that this increases the possibility of finding a triangle that is associated, especially in terms of problems that may occur, for example, spatial mismatches, chemical mismatches and / or solubility. Usually an exact repetition of a triangle with groups is unattainable, so almost similar triangles are used to ensure overlap. In some cases, triangles are chosen so that, for at least some pairs of groups on the target, both a triangle with a smaller distance between the same groups and a triangle with a large distance between the same groups are suitable for binding. This provides an overlap coefficient without repetition. Alternatively, an overlap factor of 2 or 3, lower or higher, for example, 4 or 6, and / or a fractional coefficient (for example, an average overlap value) can be used.

The overlap in the library can be uniform, or for some triangles and / or molecules, greater overlap can be achieved, for example, for molecules where there is a higher probability of spatial inconsistencies due to the framework and / or other groups, or based on experimental results that indicate that certain meters and / or triangles are difficult to communicate with.

It should be noted that if it is necessary that the molecule is deformed to form a bond,

- 2 010258 then the probability of its binding is usually lower. Thus, the actual overlap between two dissimilar triangles of the two meters can be uneven and depend on the overall probability of binding. In the general case, if the probability of finding a binding in studies is negligible, it is assumed that the meter is not connected. This helps determine the area of deformation that can be used to determine coverage and overlap. In some embodiments, implementation of the present invention, the molecules are mainly rigid, therefore, the cutoff of the degree of deformation is more clearly defined and limited.

A specific example of a method for searching for a drug substance according to an illustrative embodiment of the present invention is as follows:

(a) A library of small molecules designed to encompass all possible 3-point pharmacophores (all combinations of 3 elementary chemical groups and distances between them) is synthesized. This is the final library, which may include, for example, -100,000 compounds. It is called UBS (universal screening library) because of the generalized nature of the ability (for example, in some embodiments of the present invention) of its use for mapping a wide range of targets for which medicinal substances in the form of small molecules have been developed.

(b) For any target, UBS is screened for such a target, looking for weakly active components (affinity - 100 microM). Theoretical considerations and experimental data indicate that 100-1000 hits should be expected for any target.

(c) Carry out a numerical analysis of the active molecules, looking for:

1. 3-point pharmacophores (3PP) involved in binding of hits.

2. Reconstruction of the topography of the binding site in terms of chemical groups involved in binding. Create a complete pharmacophore (-10-20 points) of the binding site.

(b) Numerically identify molecules that can complement a fairly large (e.g., 6-8 points for nanomolar binding) subset of the complete pharmacophore. Not necessarily knowing which parts of these molecules are not directly involved in binding, they are designed in such a way as to satisfy predetermined qualities corresponding to the drug substance (for example, using the Lipinsky rule 5).

(f) Using well-known chemical data, select the molecules that are most suitable for synthesis and, based on other indicators (e.g. toxicity), and synthesize them as possible drug candidates.

(1) Conduct testing and iteration.

An aspect of some embodiments of the present invention relates to the assessment of the spatial arrangement of binding sites on a target molecule. In an illustrative embodiment of the present invention, small molecules are selected so that each of them is modeled as a set of geometric substructures that can independently bind to the target. In one example, geometric substructures can be three groups organized in a triangle. In an illustrative embodiment of the present invention, test results are analyzed to determine which of the many geometric substructures in small molecules actually binds to the target molecule. In an illustrative embodiment of the present invention, a clustering method is used to determine which geometric substructure binds by grouping together molecules that bind and which have similar geometric substructures. The result of the clustering method can be a list of all possible binding substructures. Optionally, the substructures used for analysis and for the development of meters are triangular.

In an illustrative embodiment of the present invention, an evaluation-based method is used to convert a list of geometric substructures (e.g., triangles) into complete geometric structures by:

(a) creating possible structures from the list of substructures;

(b) linking the correctness assessment to each structure; and (c) choosing between structures based on their assessment.

In an illustrative embodiment of the present invention, an estimate is the likelihood that two substructures share a part of a structure, and an optionally higher rating is given to a structure in which the part is shared as representing a structure that is more capable of binding. Alternatively or additionally, the estimate is the probability of binding of two different groups to the same binding site, and does not necessarily give a higher rating if more groups share the same binding site, since this is to minimize the pharmacophore points to the required minimum . Other heuristic rules may also be used.

In an illustrative embodiment of the present invention, the set of all potential models is not really constructed. Instead, they search for model spaces and

- 3 010258 socially build (and / or reject) models based on the definition of substructures.

In an alternative embodiment of the present invention, a clustering method is used, including, for example:

(a) creating (all) possible structures from the found triangles, optionally using specific design rules;

(b) finding the most common large substructures that are shared by multiple substructures; and (c) selecting a particular general substructure, optionally using an estimation method such as cluster size, edge size, and threshold processing of cluster size, possibly selecting the most general substructure of all those that cross a threshold. In some cases, more than one resulting final structure may be provided.

It should be noted that a real pharmacophore may not be limited by size and not be a precisely defined entity, for example, a point that is a formally external active region can act as a pharmacophore if a small molecule drug that binds to such a point includes a tail that blocks the active region from interacting with the substrate. However, often the relevance of the binding region is further away from the active region, the control region, and / or the region of conformational changes. Additionally, the protein binding affinity often decreases significantly when shifted away from such areas.

In an illustrative embodiment of the present invention, clustering structures are created in the following manner:

(a) choose a triangle as the base substructure;

(b) add a point to the base substructure if there are two triangles that, together with the triangle on the substructure, define the tetrahedron; and (c) (b) is repeated until the unused triangles for addition are completed.

An aspect of some embodiments of the present invention relates to the search for one or more molecules (for example, a drug leader) that are supposed to correspond to a target from a variety of geometric and / or chemical measurements of the target region. Measurements are not necessarily used to create a reconstructed model of the target, for which various methods of processing the model can be applied, for example, the use of suitable computer technology and software. In an illustrative embodiment of the present invention, measurements are provided by interacting the target with a plurality of measurement molecules and determining the degree of binding of the measurement molecules to the target. For example, the set of geometry of the triangles is determined by the correspondence of the meters and a correlation is found with the reconstructed three-dimensional model of the target region.

Optionally, the target region is compared with structures of known pharmaceuticals or substances similar to pharmaceuticals, for example, a library of drug leaders. Alternatively or additionally, the geometry of the target region is used to select the most suitable candidate from a relatively small number of substances. Alternatively or additionally, during the drug development process, matching is used to select or reject modifications to drug leaders that do or do not match the geometry of the target region.

In a specific example, if there is a desire to comply with the Lipinsky rules by adding or removing H-linked donors / acceptors, knowing which groups are important for binding may indicate which ones should not be removed, and knowing which parts of the molecule are not significant indicate where joins can be made without breaking the binding.

An aspect of some embodiments of the present invention relates to a library of meters for measuring a biochemical target. In an illustrative embodiment of the present invention, the library contains a large number of molecules constructed by attaching groups to a relatively small number of scaffolds. In an illustrative embodiment of the present invention, the groups are selected to have as low a molecular weight as possible. Alternatively or additionally, the library is designed to cover, in a desired manner, a set of pharmaceutically defined geometric substructures. Perhaps the geometric substructures are triangles with different groups at the vertices. In one example, the size range of the various triangles is uniformly covered.

In an illustrative embodiment of the present invention, a library is selected to provide the same (overlapping) geometric substructures based on some frameworks and / or certain molecules, for example, each substructure is provided twice or thrice. Optionally, the overlap is designed to accommodate spatial inconsistencies and / or different chemical compositions of different frameworks and / or meters.

In an illustrative embodiment of the present invention, the carcasses used include at least two, at least five, at least seven, at least 10, or any larger or medium amount, such as at least all of the following carcasses: mono-4 010258 carbon; pyrrol; quinoline pyrazinoquinazoline; isoindoloindole; isoindoloindole with an attached oxygen group; indolo [2,3-b] quinoline; pyrrolysin; 2,2'-bipyrrolone; indolysin; thiophene; Ирpyrrole; furan; benzene; pyridine; pyrimidine; pyrazine; 6H-thieno [2,3-b] pyrrole; 1,6-dihydropyrrolo [2,3b] pyrrole; 1H-indole; thieno [2,3-b] pyrimidine; 6,7-dihydropyrazolo [1,5-a] pyrimidine; quinoline; isoquinoline; quinoxaline; 3,4-dihydrobenzo [e] [1,4] diazepin-5-one; 3,8-dihydro-4H-pyrrolo [2,3-e] [1,4] diazepin-5one; 3,4-dihydrothieno [2,3-e] [1,4] diazepin-5-one; 3,6-dihydro-4H-pyrrolo [3,2-e] [1,4] diazepin-5-one; 5H, 11H-dibenzo [b, 1] [1.5] diazocin-6,12-dione; 1,4-dihydro-10H-1,4,10-1,4,10-triazabenzo [a] cyclopenta [e] cyclooctene-5,11-dione; 4H, 10H-1-thia-4,10-diazabenzo [a] cyclopenta [e] cyclooctene-5,11-dione; dipyrrolo [1,2-s; 2 ', 1'-e] imidazol-5-one; 1,4,7,9-tetrahydro-1,4,6,9-tetraazadicyclopenta [a, e] cyclooctene-5,10 dione; 4,7,9-trihydro-1-thia-4,6,9-triazadicyclopenta [a, e] cyclopentene-5,10-dione; 2,4,9-trihydro1 lambda * 4 *, 6-dithia-4,9-diazadicyclopenta [a, e] cyclooctene-5,10-dione; 6.9-dihydro-5H-1-thia5.8.9, triazacyclopenta [a] azulen-4-one; 3.10, dihydro-4H- [1,4] diazepino [5,6-b] indol-5-one; 3,6-dihydro4H- [1,4] diazepino [6,5-b] indol-5-one; 7,8-dihydro-1H-1,7,10-triazacyclohepta [e] inden-6-one; 8,9-dihydro3H-3,6,9-triazacyclohepta [e] inden-10-one; 7,8-dihydro-1H-1,5,8-triazacyclohepta [1] inden-9-one; 8.9 dihydro-5,6,9,11-tetraazacyclohepta [b] naphthalen-10-one; 3,4-dihydro [1,4] diazepino [5,6-b] quinolin-5-one;

8.9-dihydro-4,8,11-triazacyclohepta [a] naphthalen-7-one; 11H-10,11-diazabenzo [b] fluorin; α-hydroxy acids; α-amino acids; copolymer gels; bicyclo [2.2.2] octane; 2-methylene-2,3-dihydrobenzo [1,4] dioxin; 6,7-dihydro-2H-pyrazino [1,2-a] pyramidine; 9H-fluorene; 1,4-diazabicyclo [2.2.2] octane; 1-azabicyclo [2.2.2] octane; pyrrido [2,3-b] pyrimidine; 5-methylene-1,5-dihydropyrrol-2-one; benzo [4,5] imidazo [1,2-a] pyrimidine; 1,4-dihydrobenzo [4,5] imidazo [1,2-a] pyrimidine;

4.10-dihydro-1,4a, 10-triazapenantren-9-one; 1,5-dihydroimidazo [1,2-a] pyrimidin-2-one; 1,2,3,5-tetrahydroimidazo [1,2-a] pyrimidine; thiazolo [3,2-a] thieno [2,3-b] pyrimidin-5-one; 1,9-dithia-4a, 10-diazacyclopenta [b] fluoren-4-one; 5,6-dihydro-1-thia-5,7,8,9a-tetraazacyclopenta [e] azulen-4-one; 6,10 dihydro-5H-1-thia-5,7,10a-triazabenzo [e] azulen-4-one; 4,5-dihydro-3-thia-4,5a, 10-triazacyclopenta [a] fluorin; 8H-1-thiacyclopenta [a] indene; 3-thia-4,5a, 10-triazacyclopenta [a] fluorin; 6,7,9,11-tetrahydro-10thia-6,9-diazaindeno [1,2-a] azulene-5.8-dione; 2,3,6,7,12a-hexahydropyrazino [1 ', 2': 1,6] pyrido [3,4-b] indole-

1.4- dion; 5,10-dihydro-4H-2,3a, 10-triazacyclopenta [a] fluorin; 5H-pyrido [4.3-b] indole; 11H-indolisino [1,2-b] quinolin-9-one; 1,2-dihydro-2,4a, 9, triazanthracene-3,10-dione; 6H-isoindolo [2,1-a] indole;

1.5-dihydrobenzo [b] [1,4] diazepin-2-one; 5,10-dihydrodibenzo [b, e] [1,4] diazepin-11-one; 5,11-dihydrobenzo [e] pyrido [3,2-b] [1,4] diazepin-6-one; 4,9-dihydro-3-thia-4,9-diazabenzo [1] azulen-10-one; benzo [d] quinoxaline; pyrazino [2,3-b] quinoxaline; pyrido [2,1-b] quinazolin-11-one; 1-thia-4a, 9-diazacyclopenta [b] naphthalen-4-one; 2-methylene-4H-benzo [1,4] thiazin-3-one.

In the general case, a larger number of frames makes it easier to find the correct dimensions for the meters, and also deals with a wider range of spatial mismatch conditions and / or various chemical compositions. On the other hand, fewer scaffolds contribute to the uniformity of chemical behavior and synthesis methods.

In an illustrative embodiment of the present invention, the groups used include at least 2, at least 4, at least 6, or any larger number, for example, all of the following groups: Me, E1, Pr, Py, CO ₂ H, OH , N4 ^. ketone, halogen such as C1 or Br, other acids such as 8O ₃ H, PO ₃ H ₂ , and NH — C = NH (—NH ₂ ) (guanidine).

In general, using more groups can provide better accuracy in determining binding, given the possible size of the library. Synthesis methods can also be simplified using fewer groups.

An aspect of some embodiments of the present invention relates to the selection of a library of meters for use in characterizing a target. In an illustrative embodiment of the present invention, the size range of the geometry of the target is evaluated, as well as the types of bonds of the binding sites. A set of molecules that covers a range of possible sizes and types of bonds are selected from a more accessible set of molecules. The choice can be, for example, electronic with selected molecules intended for synthesis in response to the choice, or the choice is physical, with measuring molecules already available. Optionally, the assessment uses various information known about the targets. Alternatively or additionally, the assessment is performed using the first screening library, which is, for example, more flexible in the affinity of its bond types and / or uses molecules that are more flexible.

Optionally, the gauges are selected so that the library can have a significant number of repetitions, for example, to cover spatial mismatches and / or other properties of molecules that may interfere with binding. Optionally, the library includes at least one or possibly more than one multi-point linking geometry for at least some physical geometry, such as triangles and pentagons.

According to an illustrative embodiment of the present invention, such a library can be used on its own or as part of another library for various applications. In an illustrative embodiment of the present invention, such an encompassing library

- 5 010258 is used to increase the possibility of linking any of the meters in the library with the target, preferably a significant number of meters. It is noted that the standard leader library often does not provide binding at all. Optionally, the binding results are used to obtain information about the target, especially statistical information. Optionally, statistical information is used to provide structural information about the target. Optionally, the structural information contains a chemical and / or geometric structure of a significant part of the target, for example, its active region. It should be noted that in an illustrative embodiment of the present invention, even if a single binding is detected once, useful information about the target is available, and any library that helps guarantee such binding is used.

An aspect of some embodiments of the present invention relates to the development and / or creation of a library of meters for use in characterizing target molecules using geometric and / or physical measurements.

In an illustrative embodiment of the present invention, the construction of the library contains:

(a) the identification of molecules that may be suitable as meters;

(b) determining whether the identified molecules provide the required meters; and (c) confirmation that the molecules are real, for example, actually synthesized and / or having the desired chemical behavior. It should be noted that such a sequence is flexible, for example, as shown below.

In one example, such a method is used if at least a portion of the meter library is based on existing libraries. In some libraries (c) is already performed when linking the library. In addition, in one case, prior to selecting molecules, the known existing binding results of certain molecules are used as input, instead of selecting a meter and physical testing of binding affinity.

Alternatively, candidate meters may be provided as a group, for example, if a new wireframe has been added to the library. Then a large number of candidates emerge as joining various groups to the framework. However, in this case, the opposite step can be performed - the frame can be rejected, because it does not add any (or sufficient) meters that do not overlap with existing meters. Frames that create a small number of meters may be suitable for some parts of the space covered.

In an alternative method, a chemical engineering methodology is applicable for developing meters and / or frames that have the desired properties and / or geometry, for example, to fill in the missing parts of the measured space.

In an illustrative embodiment of the present invention, one or more of the following is considered as the desired properties of the meters, however, it is necessary that the meter does not have all the properties or even any of the following properties, in order to be suitable in some embodiments of the present invention:

(a) High rigidity. This may allow more accurate measurements, however, a small degree of flexibility may be appropriate to provide full coverage of the entire space. Stiffness means that the length and / or relative bond angles are not substantially changed.

(b) Low weight. This can increase the possibility of binding, even if the affinity is low, and only three points are connected on the meter.

(c) Small size. This can make it easier to measure targets and more easily avoid spatial inconsistencies.

(6) Non-toxicity. This may allow the use of meters in living cells. However, due to the different sensitivity of different cells, this often cannot be guaranteed.

(e) Good chemical behavior. This means that the meter is soluble and binds under conditions that do not deform the meter or deform by a known amount.

(1) Strong binding. This means in one embodiment of the present invention, for example, 1-100 micromoles, which is useful, for example, with low solubility or high toxicity.

In an illustrative embodiment of the present invention, one or more of the following is considered to be the desired properties of the carcasses, however, it is necessary that the carcass does not have all the properties or even any of the following properties in order to be suitable for some embodiments of the present invention:

(a) Ease of introducing groups (e.g., synthesized meters) and obtaining clear solutions of specific meters.

(b) Providing a wide range of sizes.

(c) The presence of many (e.g.> 3, better> 4,> 5) connection points. Since each hydrogen atom in the molecule is potentially an attachment point, in an illustrative embodiment of the present invention, a suitable attachment point is available for chemical

- 6 010258 manipulations.

(i) What (relatively rare in other gauges) capabilities for chemical compositions and / or gauge sizes are added to the library by including a framework.

(f) The introduction of various combinations of groups is permissible, since not all combinations will work with all frameworks.

In an illustrative embodiment of the present invention, one or more of the following is considered as desirable properties of the meter library:

(a) Coverage of the distance between bonds.

(b) Chemical coverage. Provide a wide range of groups at points at opposite ends of the ties.

(c) Coverage of substructures. For a selected substructure, for example, a triangle, all possible configurations of triangles in the target can communicate with at least one meter in the library.

(i) Compactness. Smaller libraries are better. For practical purposes, a library cannot be very small, however, usually there is no need for very large libraries.

(e) A variety of meter properties in the library to match meter coverage density, for example, lengths with less rigid bonds to cover missing or spatially distant bonds.

(1) Coating uniformity. Various types of uniformity can be provided, for example, uniformity in absolute dimensions or uniformity adjusted for chemical dependencies. For example, the distance density for short bond lengths should be greater than for long bond lengths to ensure the same normalized density for different lengths.

(e) The degree and type of overlap. Since stronger overlap is usually better for reconstruction and chemical generalization, this is often given at the cost of library size and cost. Triple overlap (for example, each triangle is provided with three meters) is an example of a compromise.

However, in general, the desired properties may depend on the target, the environment, and / or the type of search method to be used. More specifically, it is noted that in some cases, the created library is only partial, for example, covering only part of the space, suitable only for part of the target, having a lower resolution, having less (or no) overlap and / or having a disadvantage for some types of targets.

A broad aspect of some embodiments of the present invention relates to molecules, such as meters and frames, and methods for their synthesis, which can be used for libraries according to illustrative embodiments of the present invention.

Thus, according to illustrative embodiments of the present invention, there is provided a method for obtaining information about a chemically active region of a target molecule, comprising providing a set of substantially rigid chemical meters;

the implementation of the reaction of the specified target with many meters of the specified set of meters;

the study of the binding of these meters with the specified target to obtain many research results; and analyzing said research results to obtain information about said chemically active region.

Optionally, said meters allow rotation of groups of said meters. Alternatively or additionally, said meters are constructed using a rigid frame.

In an illustrative embodiment of the present invention, the constituent atoms of said meters do not move more than 1A until at least 20 kcal / M is applied to the target.

In an illustrative embodiment of the present invention, the analysis comprises identifying a plurality of spatial and chemically specific binding configurations with said active target region. Optionally, the specified configuration contains the configuration of the triangles. Alternatively or additionally, the identification comprises a configuration identification that corresponds to the configuration of the associated meter. Alternatively or additionally, the identification comprises a configuration identification that does not match the configuration of the associated meter.

Optionally, the identification comprises identification by statistical analysis of the indicated research results. Optionally, authentication comprises identification by clustering.

In an illustrative embodiment of the present invention, the identification assumes that each meter indicates a single configuration. Alternatively or additionally, the identification suggests that at least some meters

- 7 010258 denote many configurations. Alternatively or additionally, the identification comprises a classification of meters by chemical groups at the tops of said configurations.

In an illustrative embodiment of the present invention, the method comprises reconstructing a spatial map of at least a portion of said chemically active region from at least two of the indicated research results, said portion comprising at least four chemical binding regions. Optionally, said portion includes at least six chemical binding regions.

In an illustrative embodiment of the present invention, the method comprises reconstructing a spatial map of at least a portion of said chemically active region from at least two configurations, said portion comprising at least four chemical binding points. Optionally, said portion includes at least six chemical binding regions.

In an illustrative embodiment of the present invention, the reconstruction comprises: a test reconstruction of a plurality of spatial maps from said configurations;

assessment of these cards; and selecting a spatial map based on its rating.

Alternatively or additionally, the reconstruction comprises: a test reconstruction of a plurality of spatial maps from the indicated configurations; clustering of these cards according to general structures; and selecting a spatial map based on the relative property of the cluster to which it belongs.

Optional specified comparison property contains size.

In an illustrative embodiment of the present invention, the specified spatial map includes enough binding points to ensure the binding of the drug substance in the form of a small molecule having a chemical profile corresponding to the binding points. Optionally, the specified spatial map includes at least 6 points of binding. Optionally, the specified spatial map includes at least 8 points of binding.

In an illustrative embodiment of the present invention, the specified set of meters contains a set of meters with at least 10,000 meters. Optionally, said meter set comprises a meter set with at least 50,000 meters.

In an illustrative embodiment of the present invention, said meters comprise groups arranged in spatial configurations, where said meters are selected to cover the virtual space of spatial chemical configurations.

In an illustrative embodiment of the present invention, essentially every point in the virtual space that is covered by said meters is covered by at least two meters. Optionally, essentially every point in the virtual space that is covered by said meters is covered by at least three meters.

In an illustrative embodiment of the present invention, at least 0.5% of said meters is associated with said target. Optionally, 1% of said meters binds to said target. Optionally, 3% of said meters binds to said target.

In an illustrative embodiment of the present invention, at least 50% of said meters are determined by adding groups to a set of less than 100 scaffolds. Optionally, 50% of these meters are determined by adding groups to a set of less than 50 scaffolds.

In an illustrative embodiment of the present invention, at least said set of meters uses less than 15 different chemical groups to determine the chemical behavior of said meters.

In an illustrative embodiment of the present invention, at least said set of meters uses less than 10 different chemical groups to determine the chemical behavior of said meters.

In an illustrative embodiment of the present invention, said study is a functional study. Alternatively or additionally, said study is a binding study. Alternatively or additionally, said assay is a cell assay. Alternatively or additionally, said study is a flow study.

In an illustrative embodiment of the present invention, said functional assay is performed in the presence of a natural substrate of said target.

In an illustrative embodiment of the present invention, said target comprises a protein comprising a biochemically active region adapted to bind to a substrate. Optionally, said chemically active region comprises a region comprising a biochemically active region. Alternatively or additionally, said chemically active region comprises

- 8 010258 control region of the specified protein.

In an illustrative embodiment of the present invention, the analysis comprises an analysis of successful binding of at least 60 meters. Alternatively or additionally, the analysis comprises an analysis of the successful binding of at least 10 meters. Alternatively or additionally, the analysis comprises an analysis of the successful binding of at least 100 meters.

In an illustrative embodiment of the present invention, the identification comprises identifying at least 40 different configurations. Alternatively or additionally, the identification comprises the identification of at least 10 different configurations. Alternatively or additionally, the identification comprises the identification of at least 100 different configurations.

In an illustrative embodiment of the present invention, the method comprises comparing said map of a leader database; and selecting a leader from said database for further use, sensitive to similarities or lack of similarities between said leader and said map.

Alternatively or additionally, the method comprises comparing said map of a leader database; and the deviation of the leader from the specified database from further use, sensitive to the similarities between the specified leader and the specified card.

Alternatively or additionally, the method comprises constructing a leader having similarities with said map. Optionally, the construction comprises a construction using said meters or frames used to define said meters.

In an illustrative embodiment of the present invention, the method comprises comparing said configurations with a leader database; and selecting a leader from said database for future use that is sensitive to matching said configurations with said leader.

In an illustrative embodiment of the present invention, the method comprises constructing a leader based on said configurations.

In an illustrative embodiment of the present invention, the method comprises selecting at least one of said meters as a leader in the search for a drug substance.

In an illustrative embodiment of the present invention, the method comprises comparing the binding of meters with a similar binding geometry to obtain spatial inconsistency data; and analyzing spatial inconsistency data to provide geometric information about the specified target.

According to an exemplary embodiment of the present invention, there is also provided a method for identifying the presence of multiple spatial chemical configurations in a target, comprising examining the target with a plurality of meters having known spatial chemical configurations at their vertices to provide multiple research results;

definition of an array of spaces, with one space for each set of chemical behavior of the vertices of each configuration;

indicating the indicated results according to the indicated spaces for creating the clusters; and identifying the presence of a configuration in the indicated target from the indicated clusters. Optionally, the indication contains the distribution of the indication sensitive to the distribution function. Optionally, said propagation function is dependent on the estimated binding energy of the meter to said target.

According to an exemplary embodiment of the present invention, there is also provided a method for reconstructing a spatial form of a chemical binding configuration of a target from a set of subforms, each of which denotes a portion of said binding configuration, comprising selecting a base from said subforms;

the selection of at least two subforms having a property in which they correspond to each other at least along one of their sides and correspond to the specified base along their other side;

the accumulation of these subforms for the specified base and the repetition of the specified selection and the specified accumulation until then, until all these subforms are used or not used, thus providing a form of communication configuration of the specified target. Optionally, the method of variationally comprises repeating said selection, accumulation and repetition using different order of selection of subforms. Optionally, the method comprises repeating said base selection and said changing recurring selection for a variety of different types of base selection. Optionally, the method comprises clustering a plurality of such forms according to forms shared by subcomponents. Optionally, the method comprises selecting a form of subcomponents as the resulting form based on said clustering

- 9,010,258.

In an illustrative embodiment of the present invention, said subforms comprise triangles. Alternatively or additionally, said subforms define chemical behavior at their peaks, whereby it is said that the two sides correspond if chemical behavior at their peaks corresponds.

In an illustrative embodiment of the present invention, two sides are indicated as corresponding if their lengths are similar.

According to an exemplary embodiment of the present invention, there is also provided a method for selecting a scaffold for use in creating part of a screening library, comprising providing a potential scaffold molecule comprising a set of possible group attachment points;

determining the rigidity of the molecule and the deviation of the specified potential frame molecule, sensitive to the lack of rigidity of the specified frame. Optionally, said stiffness disadvantage is absolute. Alternatively, said lack of stiffness is relative to other potential frameworks.

In an illustrative embodiment of the present invention, the method comprises selecting a framework based on the number of rings thereof.

In an illustrative embodiment of the present invention, the method comprises determining a plurality of measurement molecules that can be created by adding groups to said potential scaffold molecule;

determination of the existing part of the library, the spatial chemical configurations of which are added using the indicated molecules, and the selection of the indicated potential framework molecule, if one or more significant spatial chemical configurations can be added to the indicated part of the library. Optionally, the method comprises selecting a chassis based on the number of configurations added by the chassis. Alternative or additionally, these significant spatial configurations are configurations not previously provided or not overlapping.

According to an exemplary embodiment of the present invention, there is also provided a method for selecting a measurement molecule to add to a screening library, comprising providing a set of chemical molecules and at least a portion of a screening library; selection of a potential measuring molecule from a specified set of chemical molecules; determining the stiffness of the specified potential measuring molecule and the deviation of the specified potential measuring molecule, sensitive to the lack of rigidity of the specified measuring molecule. Optionally, said stiffness disadvantage is absolute. Alternatively, said lack of stiffness is relative to other potential frameworks.

In an illustrative embodiment of the present invention, the method includes determining coverage, in the space of chemical configurations, of a specified portion of the screening library;

determining at least one spatial chemical configuration of said potential molecule; and selecting said potential measuring molecule if it adds at least one significant spatial chemical configuration to said screening library.

Optionally, providing a set of molecules comprises creating said molecules using a single framework to which groups are selectively attached. Alternatively or additionally, providing a set of molecules comprises providing a chemical library.

In an illustrative embodiment of the present invention, said meter is selected if it adds at least one spatial chemical configuration not previously provided or overlapping the provided configuration.

According to an illustrative embodiment of the present invention, there is also provided a method for creating at least a portion of a screening library, comprising selecting a framework molecule to which groups can be added;

determination of the set of potential meters that can be created by attaching groups to the specified frame; and selecting a subset of these meters that do not substantially overlap in chemical configurations. Optionally, the method comprises deflecting potential meters that add more than six spatial chemical configurations.

According to an exemplary embodiment of the present invention, there is also provided a method for reducing a screening library, comprising, for each molecule in at least a portion of said library, determining substantially all spatial chemical configurations of a number of binding points provided by the molecule; and

- 10 010258 removal of many molecules that add redundant spatial chemical configurations. Optionally, the specified specific number is three.

According to an illustrative embodiment of the present invention, there is also provided a method for reducing a screening library, comprising for each molecule in at least a portion of said library calculating the probability of binding of said molecules from an energy point of view and removing at least some molecules whose probability of binding is below a threshold value. Optionally, the indicated binding probability is calculated using a formula that is inversely related to the flexibility of the molecule. Alternatively or additionally, the indicated binding probability is evaluated at least based on the solubility of the molecule.

According to an exemplary embodiment of the present invention, there is also provided a method for developing a screening library for a target target molecule, comprising determining a desired range of distances between binding points for direct identification with said library;

determining the desired overlap between the units provided by the measuring molecules of the specified library;

Defining a set of desired link types in such a way that they differ from each other; and creating a plurality of gauges, said gauges, each defining a variety of binding types and distances between them, so that said gauges cover a space of spatial chemical configurations that includes said distances and said binding types with said desired overlap. Optionally creating multiple groups comprises creating by attaching groups to wireframes. Alternatively or additionally, said gauges cover the space of spatial chemical configurations of triangles of binding points. Alternatively or additionally, said objective of the target molecule to be designed contains proteins.

In an illustrative embodiment of the present invention, said number of overlaps is at least two. Alternatively, the indicated number of overlaps is at least four. Alternatively, the indicated number of overlaps is at least six.

In an illustrative embodiment of the present invention, said meters are substantially rigid. Alternatively or additionally, said coating allows for the natural flexibility of binding.

In an illustrative embodiment of the present invention, the creation comprises creating substantially the same configurations using different meters, thereby providing at least a portion of said overlap. Optionally, the creation comprises providing a repetition factor of at least two.

In an illustrative embodiment of the present invention, the creation comprises the creation of essentially different configurations using different meters, different configurations of which overlap due to their degree of flexibility, thereby providing at least a portion of said overlap.

In an illustrative embodiment of the present invention, the method comprises creating a set of drug leaders for said target based on said information. Optionally, the method comprises removing known drug leaders for said target from said kit.

According to an illustrative embodiment of the present invention also provides a set of leaders created using one of the methods described above.

According to an illustrative embodiment of the present invention, there is also provided a drug leader comprising a plurality of rigid frame sections of a molecule;

at least one compound interacting with said frame sections of the molecule; and a plurality of groups attached to said frames.

According to an exemplary embodiment of the present invention, there is also provided a screening library containing at least 10,000 molecules created by attaching groups to a set of less than 50 framework molecules. Optionally, less than 20 framework molecules are used to create said at least 10,000 molecules. Alternatively or additionally, said scaffolds include at least one of the following scaffold molecules: thiophene; 1H-pyrrole; furan; benzene; pyridine; pyrimidine; pyrazine; 6H-thieno [2,3-b] pyrrole; 1,6-dihydropyrrolo [2,3-b] pyrrole; 1H-indole; thieno [2.36] pyrimidine; 6,7-dihydropyrazolo [1,5-a] pyrimidine; quinoline; isoquinoline; quinoxaline; 3,4-dihydrobenzo [e] [1,4] diazepin-5-one; 3,8-dihydro-4H-pyrrolo [2,3-e] [1,4] diazepin-5-one; 3,4-dihydro-11 010258 thieno [2,3-e] [1,4] diazepin-5-one; 3,6-dihydro-4H-pyrrolo [3,2-e] [1,4] diazepin-5-one; 5H, 11Ndibenzo [b, 1] [1.5] diazocin-6,12-dione; 1,4-dihydro-10H-1,4,10-1,4,10-triazabenzo [a] cyclopenta [e] cyclooctene-5.11-dione; 4H, 10H-1-thia-4,10-diazabenzo [a] cyclopenta [e] cyclooctene-5,11-dione; dipyrrolo [1,2s; 2 ', 1'-e] imidazol-5-one; 1,4,7,9-tetrahydro-1,4,6,9-tetraazadicyclopenta [a, e] cyclooctene-5,10-dione; 4,7,9trihydro-1-thia-4,6,9-triazadicyclopenta [a, e] cyclopentene-5,10-dione; 2,4,9-trihydro-1 lambda * 4 *, 6-dithia-4,9-diazadicyclopenta [a, e] cyclooctene-5,10-dione; 6,9-dihydro-5H-1-thia-5,8,9-triazacyclopenta [a] azulen-4-one; 3,10-dihydro-4H- [1,4] diazepino [5,6-b] indol-5-one; 3,6-dihydro-4H- [1,4] diazepino [6,5b] indol-5-one; 7,8-dihydro-1H-1,7,10-triazacyclohepta [e] inden-6-one; 8,9-dihydro-3H-3,6,9triazacyclohepta [e] inden-10-one; 7,8-dihydro-1H-1,5,8-triazacyclohepta [1] inden-9-one; 8,9-dihydro

5.6.9.11 - tetraazacyclohepta [b] naphthalene-10-one; 3,4-dihydro [1,4] diazepino [5,6-b] quinolin-5-one; 8,9-dihydro-4,8,11-triazacyclohepta [a] naphthalen-7-one; 11H-10,11-diazabenzo [b] fluorin; α-hydroxy acids; α-amino acids; copolymer gels; bicyclo [2.2.2] octane; 2-methylene-2,3-dihydrobenzo [1,4] dioxin; 6,7-dihydro-2H-pyrazino [1,2-a] pyramidine; 9H-fluorene; 1,4-diazabicyclo [2.2.2] octane; 1-azabicyclo [2.2.2] octane; pyrrido [2,3-th] pyrimidine; 5-methylene-1,5-dihydropyrrole-2one; benzo [4,5] imidazo [1,2-a] pyrimidine; 1,4-dihydrobenzo [4,5] imidazo [1,2-a] pyrimidine; 4,10-dihydro1,4a, 10-triazapenantren-9-one; 1,5-dihydroimidazo [1,2-a] pyrimidin-2-one; 1,2,3,5-tetrahydroimidazo [1,2-

a] pyrimidine; thiazolo [3,2-a] thieno [2,3-th] pyrimidin-5-one; 1,9-dithia-4a, 10-diazacyclopenta [b] fluoren-4-one; 5,6-dihydro-1-thia-5,7,8,9a-tetraazacyclopenta [e] azulen-4-one; 6,10-dihydro-5H-1-thia-5,7,10atriazabenzo [e] azulen-4-one; 4,5-dihydro-3-thia-4,5a, 10-triazacyclopenta [a] fluorin; 8H-1-thiacyclopenta [a] indene; 3-thia-4,5a, 10-triazacyclopenta [a] fluorin; 6,7,9,11-tetrahydro-10-thia-6,9-diazaindeno [1,2-a] azulene-5.8-dione; 2,3,6,7,12a-hexahydropyrazino [1 ', 2': 1,6] pyrido [3,4-b] indole-1,4-dione; 5.10 dihydro-4H-2,3a, 10-triazacyclopenta [a] fluorin; 5H-pyrido [4.3-b] indole; 11H-indolisino [1,2-b] quinolin-9-one; 1,2-dihydro-2,4a, 9, triazanthracene-3,10-dione; 6H-isoindolo [2,1-a] indole; 1,5-dihydrobenzo [b] [1,4] diazepin-2-one; 5,10-dihydrodibenzo [b, e] [1,4] diazepin-11-one; 5,11-dihydrobenzo [e] pyrido [3,2-b] [1,4] diazepin-6-one; 4,9-dihydro-3-thia-4,9-diazabenzo [1] azulen-10-one; benzo [d] quinoxaline; pyrazino [2,3-b] quinoxaline; pyrido [2,1-b] quinazolin-11-one; 1-thia-4a, 9-diazacyclopenta [b] naphthalen-4-one; 2-methylene-4H-benzo [1,4] thiazin-3-one.

In an illustrative embodiment of the present invention, at least 4 of these frames have exactly one ring. Alternatively or additionally, at least 4 of these frames have exactly two rings. Alternatively or additionally, at least 4 of these frames have exactly three rings. Alternatively or additionally, at least 4 of these frames have exactly four rings. Alternatively or additionally, said library includes at least 50,000 molecules thus created. Alternatively or additionally, said library includes at least 100,000 molecules thus created.

In an illustrative embodiment of the present invention, said scaffolds include at least three of the following scaffold molecules. Alternatively or additionally, said scaffolds include at least ten of the following scaffold molecules as indicated below.

In an illustrative embodiment of the present invention, these created molecules are essentially rigid. Alternatively or additionally, these molecules cover the space of configurations of spatial geometric patterns of types of binding points, comprising at least 25% of the patterns that exist in protein targets. Optionally, these molecules comprise at least 50% of the patterns.

In an illustrative embodiment of the present invention, said molecules span a space defining at least 4 distinct chemical types of binding points.

In an illustrative embodiment of the present invention, said molecules span a space defining at least 5 distinct chemical types of binding points.

According to an exemplary embodiment of the present invention, there is also provided a screening library containing at least 100 measuring molecules created by attaching groups to at least one of the following scaffolds: thiophene; 1H-pyrrole; furan; benzene; pyridine; pyrimidine; pyrazine; 6H-thieno [2,3-b] pyrrole; 1,6-dihydropyrrolo [2,3-b] pyrrole; 1H-indole; thieno [2,3-th] pyrimidine; 6,7 dihydropyrazolo [1,5-a] pyrimidine; quinoline; isoquinoline; quinoxaline; 3,4-dihydrobenzo [e] [1,4] diazepin-5-one; 3,8-dihydro-4H-pyrrolo [2,3-e] [1,4] diazepin-5-one; 3,4-dihydrothieno [2,3e] [1,4] diazepin-5-one; 3,6-dihydro-4H-pyrrolo [3,2-e] [1,4] diazepin-5-one; 5H, 11H-dibenzo [b, 1] [1.5] diazocin-6,12-dione; 1,4-dihydro-10H-1,4,10-1,4,10-triazabenzo [a] cyclopenta [e] cyclooctene-

5.11- dion; 4H, 10H-1-thia-4,10-diazabenzo [a] cyclopenta [e] cyclooctene-5,11-dione; dipyrrolo [1,2-s; 2 ', 1'-

e] imidazol-5-one; 1,4,7,9-tetrahydro-1,4,6,9-tetraazadicyclopenta [a, e] cyclooctene-5,10-dione; 4,7,9trihydro-1-thia-4,6,9-triazadicyclopenta [a, e] cyclopentene-5,10-dione; 2,4,9-trihydro-1 lambda * 4 *, 6dithia-4,9-diazadicyclopenta [a, e] cyclooctene-5,10-dione; 6,9-dihydro-5H-1-thia-5,8,9-triazacyclopenta [a] azulen-4-one; 3,10-dihydro-4H- [1,4] diazepino [5,6-b] indol-5-one; 3,6-dihydro-4H- [1,4] diazepino [6,5-

b] indol-5-one; 7,8-dihydro-1H-1,7,10-triazacyclohepta [e] inden-6-one; 8,9-dihydro-3H-3,6,9-triase-12 010258 cyclohepta [e] inden-10-one; 7,8-dihydro-1H-1,5,8-triazacyclohepta [1] inden-9-one; 8,9-dihydro-5,6,9,11 tetraazacyclohepta [b] naphthalen-10-one; 3,4-dihydro [1,4] diazepino [5,6-b] quinolin-5-one; 8,9-dihydro

4,8,11-triazacyclohepta [a] naphthalen-7-one; 11H-10,11-diazabenzo [b] fluorin; α-hydroxy acids; α-amino acids; copolymer gels; bicyclo [2.2.2] octane; 2-methylene-2,3-dihydrobenzo [1,4] dioxin; 6,7 dihydro-2H-pyrazino [1,2-a] pyramidine; 9H-fluorene; 1,4-diazabicyclo [2.2.2] octane; 1-azabicyclo [2.2.2] octane; pyrido [2,3-4] pyrimidine; 5-methylene-1,5-dihydropyrrol-2-one; benzo [4,5] imidazo [1,2a] pyrimidine; 1,4-dihydrobenzo [4,5] imidazo [1,2-a] pyrimidine; 4,10-dihydro-1,4a, 10-triazapenantren-9one; 1,5-dihydroimidazo [1,2-a] pyrimidin-2-one; 1,2,3,5-tetrahydroimidazo [1,2-a] pyrimidine; thiazolo [3,2-

a] thieno [2,3-4] pyrimidin-5-one; 1,9-dithia-4a, 10-diazacyclopenta [b] fluoren-4-one; 5,6-dihydro-1-thia5,7,8,9a-tetraazacyclopenta [e] azulen-4-one; 6,10-dihydro-5H-1-thia-5,7,10a-triazabenzo [e] azulen-4-one;

4,5-dihydro-3-thia-4,5a, 10-triazacyclopenta [a] fluorin; 8H-1-thiacyclopenta [a] indene; 3-thia-4,5a, 10 triazacyclopenta [a] fluorin; 6,7,9,11-tetrahydro-10-thia-6,9-diazaindeno [1,2-a] azulene-5.8-dione;

2,3,6,7,12a-hexahydropyrazino [1 ', 2': 1,6] pyrido [3,4-b] indole-1,4-dione; 5,10-dihydro-4H-2,3a, 10-triazacyclopenta [a] fluorin; 5H-pyrido [4.3-b] indole; 11H-indolisino [1,2-b] quinolin-9-one; 1,2-dihydro 2,4,4,9-triazanthracene-3,10-dione; 6H-isoindolo [2,1-a] indole; 1,5-dihydrobenzo [b] [1,4] diazepin-2-one;

5,10-dihydrodibenzo [b, e] [1,4] diazepin-11-one; 5,11-dihydrobenzo [e] pyrido [3,2-b] [1,4] diazepin-6-one; 4,9-dihydro-3-thia-4,9-diazabenzo [1] azulen-10-one; benzo [d] quinoxaline; pyrazino [2,3-b] quinoxaline; pyrido [2,1-b] quinazolin-11-one; 1-thia-4a, 9-diazacyclopenta [b] naphthalen-4-one; 2-methylene-4H-benzo [1,4] thiazin3-one.

Optionally, these molecules are created using at least one of the following scaffolds: thiophene; 1H-pyrrole; furan; benzene; pyridine; pyrimidine; pyrazine; 6H-thieno [2,3-

b] pyrrole; 1,6-dihydropyrrolo [2,3-b] pyrrole; 1H-indole; thieno [2,3-4] pyrimidine; 6,7-dihydropyrazolo [1,5-a] pyrimidine; quinoline; isoquinoline; quinoxaline; 3,4-dihydrobenzo [e] [1,4] diazepin-5-one; 3,8-dihydro-4H-pyrrolo [2,3-e] [1,4] diazepin-5-one; 3,4-dihydrothieno [2,3-e] [1,4] diazepin-5-one; 3,6-dihydro-4H-pyrrolo [3,2-e] [1,4] diazepin-5-one; 5H, 11H-dibenzo [b, 1] [1.5] diazocin-6,12-dione; 1,4-dihydro10H-1,4,10-1,4,10-triazabenzo [a] cyclopenta [e] cyclooctene-5,11-dione; 4H, 10H-1-thia-4,10-diazabenzo [a] cyclopenta [e] cyclooctene-5,11-dione; dipyrrolo [1,2-e; 2 ', 1'-e] imidazol-5-one.

In an illustrative embodiment of the present invention, said at least 100 molecules comprise at least 300 molecules. Alternatively or additionally, said at least 100 molecules of said library are created using a single indicated framework molecule.

According to an exemplary embodiment of the present invention, a screening library is also provided comprising a set of at least 10,000 substantially rigid molecules. Optionally, said kit contains at least 50,000 substantially rigid molecules. Alternatively or additionally, said kit contains at least 100,000 substantially rigid molecules.

In an illustrative embodiment of the present invention, said kit is selected so as to generally have an expected binding ratio of at least 0.1% of the protein target library. Optionally, the expected binding ratio is at least 0.5%.

In an illustrative embodiment of the present invention, said kit is designed to provide molecules with a uniform probability of hits for a generalized target in a 1: 100 ratio for the entire kit. Optionally, said ratio is 1:10.

In an illustrative embodiment of the present invention, said set covers a space of spatial chemical configurations, each such configuration defining a plurality of binding points having distances between them, the set covering essentially all possible configurations in space in a given distance range.

According to an exemplary embodiment of the present invention, there is also provided a screening library containing a plurality of at least 5,000 measuring molecules, each of which defines at least one spatial configuration of points of binding type in which essentially every point in the space of such configurations is covered by at least two different measuring molecules. Optionally, each point is covered by at least two essentially identical spatial configurations. Alternatively or additionally, each point is covered by at least two essentially different spatial configurations. Alternatively or additionally, said space is the space of triangles defined by the type of binding at the vertices and the distances between the vertices. Optionally, the specified space includes distances: between 4 and 8A (angstrom = 10 ^-10 m). Alternatively or additionally, said space includes distances from 2A to 10A. Alternatively or additionally, said space includes at least 5 different types of binding. Optionally, said space includes at least 7 different types of binding.

In an illustrative embodiment of the present invention, the specified space

- 13 010258 includes directional binding types. Alternatively or additionally, said space includes directional binding types.

In an illustrative embodiment of the present invention, substantially each of said points in said space is covered by at least three meters.

In an illustrative embodiment of the present invention, substantially all meters include multiple configurations of the specified space.

According to an exemplary embodiment of the present invention, there is also provided a method for acquiring information about the binding behavior of a target molecule, comprising providing a set of substantially rigid chemical meters, it being contemplated that a significant number of said meters are bound to said target;

the implementation of the reaction of the specified target with multiple meters of the specified set of meters and physical analysis of the structure of the specified target associated with the meter. Optionally, the physical analysis comprises NMR analysis. Alternatively or additionally, the physical analysis comprises analysis using x-ray crystallography. Alternatively or additionally, the physical analysis comprises analysis using binding to a set of meters. Alternatively or additionally, the method essentially comprises superimposing a plurality of structures obtained using said physical analysis.

According to an illustrative embodiment of the present invention also provides a method of constructing a leader, comprising providing a set of essentially rigid chemical meters;

the implementation of the reaction of the specified target with many meters of the specified set of meters; and the study of the binding of these meters with the specified target to obtain many research results; and designing a leader based on the indicated research results. Optionally, constructing a leader comprises connecting a plurality of meters together for which it was found in the study that they bind. Alternatively or additionally, the construction of the leader contains a modification of the existing molecules to have groups that correspond to the found binding sites using this study.

Brief Description of the Drawings

Non-limiting embodiments of the present invention will be described with reference to the following description of illustrative embodiments in conjunction with the drawings. The drawings, as a rule, do not reflect the scale, and any measurements are intended for illustrative purposes only and not for limitation. In the drawings, identical structures, elements or parts that are shown in more than one drawing are preferably denoted by the same or similar reference numeral in all the drawings in which they are shown, in which FIG. 1 is a schematic diagram of a protein target including a plurality of binding points;

FIG. 2 is a flowchart of a method for searching for a drug substance according to an illustrative embodiment of the present invention;

FIG. 3 is a flowchart of a method for measuring a target according to an illustrative embodiment of the present invention;

FIG. 4A schematically shows an illustrative meter according to an illustrative embodiment of the present invention;

in FIG. 4B shows the meter of FIG. 4A interacting with the protein target of FIG. one;

FIG. 5 is a flowchart of a method for determining which triangles actually bind to a target according to an illustrative embodiment of the present invention;

FIG. 6A is a flowchart of a method for determining a spatial arrangement for binding sites according to the results of the method of FIG. 5 according to an illustrative embodiment of the present invention;

FIG. 6B is a flowchart of an alternative method for determining the spatial arrangement for binding sites from the results of the method of FIG. 5 according to an illustrative embodiment of the present invention.

Similar Description of Exemplary Embodiments

1. Overview

2. Illustrative method of finding a drug substance

3. Details of the method

3.1 Target Measurement

4. Illustrative studies

4.1 Functional research

4.2 Binding Studies

- 14 010258

5. Meters, general view

5.1 Illustrative meter

5.2 Number of groups per unit of measure

5.3 Number of groups in the meter

5.4 Group types

5.5 Overlapping units in a set

6. Reconstruction

6.1 Selecting a triangle

6.2 Reconstructing a configuration layout

6.3 Reconstruction options

6.4 Alternative reconstruction methods

7. Analysis

7.1 Overview

7.2 Verification of reconstruction

7.3 Binding Strength

7.4 Interactions within relationships

7.5 Geometric analysis

7.6 Determination of spatial discrepancies

7.7 Identification of control areas

7.8 Other types of map analysis

8. The use in the methods of searching for medicinal substances

8.1 Overview

8.2 Creating a drug

8.3 Creating a Leader

8.4 Leader Description

8.5 Leader Search

8.6 Leader rejection

8.7 Target Mapping

8.8 Checking target suitability

8.9 Target Sectioning

8.10 Analysis and improvement of the drug substance and leader

8.11 Drug selection

8.12 Drug Improvement

8.13 Failure to analyze and re-develop a drug substance

8.14 Additional analysis related to drug discovery

8.15 Directional drug search method

8.16 Creating a convenient model

9. Illustrative Search Applications

9.1 Overview

9.2 Screening drug development

9.3 Screening alternative drug development

9.4 Development of a drug substance based on the structure

9.5 Modular assembly of ligands

10. Illustrative non-search applications

11. Use of known information

12. Iterative measurement

Meters, physical properties

13.1 Overview

13.2 Wireframe

13.3 Volumetric geometry of meters

13.4 Flexibility

13.5 Meter lengths

13.6 Environmental Stability

13.7 Uniqueness of meters and overlapping sides and triangles

13.8 Weight and size of meters

14. Specific and general development of a set of meters

14.1 Example Coverage Library Size

14.2 Selecting a subset of meters

14.3 Development of a library of meters

14.4 Method of building libraries

14.5 Wireframe selection method

- 15 010258

14.6 Meter selection method

14.7 Meter Synthesis

14.8 Developing a Mixed Library

14.9 Ensuring library reliability

14.10 The human factor during library development

15. Experiments and examples

15.1 Experiment 1

15.2 Experiment 2

16. The book of synthesis

16.1 Framework of the 6-membered ring of benzenes, pyrimidines

16.2 6,6,5,6-cyclic framework of indolo [2,3-quinoline]

16.3 6,5,5,6-tetracyclic frameworks of isoindoloindoles and isoindoloindolones 16.3.1 Isoindoloindolones

16.4 Monoatomic framework

16.5 6,7-bicyclic framework of benzodiazepines

16.6 6,6,6-tricyclic pyrazinoquinosalinone framework

16.7 Frame 5-membered pyrrole ring

16.8 Thiophenes and related scaffolds

16.8.1 5,5-bicyclic frameworks

16.8.2 5,6-bicyclic frameworks

16.8.3 5,8,5 and 5,8,6-tricyclic and 5,5,8,6 and 5,5,8,5-tetracyclic frames

16.8.4 5,7-bicyclic framework

16.8.5 5,6,5,6-tetracyclic and 5,6,5-tricyclic frameworks

16.8.6 5-6-5-6-tetracyclic framework

16.8.7 5-6-5-tricyclic framework

1. Overview

The high specificity of many biological molecules, such as enzymes, is due to the presence, for example, in such molecules, specifically spatially located binding sites. It is assumed that the substrate molecule for successful interaction with the enzyme must correspond (at least in part) to a specific spatial location. In the pharmaceutical industry, this specificity can be used by searching for small molecules that mimic the shape or chemical affinity of the substrate molecule. In conventional drug discovery methods, such small molecules are found by testing millions of small molecules, and after finding a molecule that exhibits some affinity, fine-tune it chemically until a better binding is found. In an illustrative embodiment of the present invention, a particular spatial arrangement is mapped and such a map is used to facilitate the search for a drug substance and, of course, to search for new suitable drug substances in the form of small molecules. It should be noted that in the general case, the spatial geometry of the binding sites is three-dimensional.

Further in the description, the molecule is called the target, and the spatial arrangement is called the target region or pharmacophore. However, as will be apparent, the mapping method according to an illustrative embodiment of the present invention and / or its derivatives are useful in addition to drug discovery, for example, in the development of herbicides and target antibodies. Thus, the terms used are used for convenience, and not to limit the desired scope, unless otherwise indicated.

FIG. 1 is a schematic diagram of a protein target 100 including a plurality of binding sites 102 (and 108). As shown, binding sites 102 are located in the region of target 104, which is designed to bind to a protein substrate. In some proteins, the target region of interest is the control region of protein 106 (with binding sites 108), which upon binding changes the behavior of the protein (for example, changing the configuration of the receiving region of the protein substrate). Perhaps many non-functional binding sites 110 are found on the outside of the protein.

Although the following description focuses on finding small molecules to target enzymatic proteins, target 100 can be any biomolecule whose biological activity can be influenced in a desired way by binding the molecule to it. For example, target 100 may be one or more DNA, RNA, signaling proteins such as hormones, structural hormones, growth factors, other proteins, antibodies, cell receptors, ion channels, cytokines, complexes, membranes, toxins (biological and synthetic) , medicinal substances in the form of small and large molecules and hydrocarbons. Non-biological use is also provided, for example, for the study of enzymes used as detergents and industrial use. Additionally, the desired molecules should not be small molecules for some applications, for example, it may be a peptide, protein, antibody or metal complex.

- 16 010258

According to exemplary embodiments of the present invention, mapping of the target region 104 (or 106) is accomplished by creating a plurality of geometric and / or chemical affinity measurements of the target region and then correlating the measurements to provide a three-dimensional model of the target region 104. In an illustrative embodiment of the present invention, measurements are made using a set of defined measuring molecules. In an illustrative embodiment of the present invention, the meters are selected for some association of geometric and / or chemical affinity with an optionally small range of flexibility. In a set of meters, a large range of geometry, dimensions, and / or affinity is optionally provided using a large number of specific meters.

In an illustrative embodiment of the present invention, each measurement molecule creates multiple measurements at the same time, and there is overlap between measurements created by different measurement molecules. Optionally, a processing step is provided in which a complex measurement is interconnected with meters to obtain indications of individual measurements, which are then used to reconstruct a three-dimensional map. Additional collateral information is not necessarily used for processing and / or for analysis and / or for using processing results. Various examples of such secondary information are described below.

2. Illustrative method of finding a drug substance

FIG. 2 is a flowchart of a method for searching for a drug substance 200 according to an illustrative embodiment of the present invention. At step 202, a target 100 is provided for which a drug substance is to be developed. Optionally, in step 204, a subset of meters is selected to measure the target 100. Alternatively, one set of meters is used for all targets.

At step 206, meters are used to measure the spatial arrangement of the interaction sites 102 and / or 108.

At step 208, a model of at least a portion of the active and / or control regions of the target 100 is reconstructed from the measurements. At step 210 and 212, one or more molecules that correspond to the measurements are determined. At step 214, the corresponding molecules are further processed to provide a drug substance.

Further details of this method are described below. Alternative methods are also described below.

3. Details of the method

3.1 Target Measurement

FIG. 3 is a flowchart of a method for measuring a target 300 according to an illustrative embodiment of the present invention. At step 302, a certain amount of target 100 and one or more meters are combined into a container and possibly left for incubation (304) so that the meters can contact the sites of interaction on target 100. In some embodiments, the target is also incubated with a substrate or another molecule. Such incubation can be provided for various reasons, for example, to improve the conformational change of the target to facilitate dissolution, to maintain the target in an active state and / or part of a functional analysis. The target may be in a relatively clean state, for example, a purified segment of replicated DNA. Alternatively, the target may be provided in a more natural environment, for example, in a living cell or associated with molecules (for example, whose interaction effects may not be known). Optionally, a plurality of overlapping meters (i.e., overlapping them is possible to measure the same or similar spatial geometry) are incubated together in the same study.

At step 306, the degree of binding of the meter to the target 100 is optionally determined. The method used may depend on the type of study used, various examples of which are provided below. Alternatively or additionally, in step 308, the effect on the function of the target 100 is determined, various examples are provided below.

It should be noted that studies suitable for searching for binding of a test molecule to a target molecule are well known for drug discovery and many are suitable for the present invention, possibly without modification.

The research process can then be repeated with another meter and / or in other conditions (310), such as solvent, temperature and pH. Various conditions may be used, for example, to determine the binding strength and / or to compensate for unavailable gauges, for example, by stimulating conformational changes on the target 100. Repetition may depend on preliminary measurements and / or measurement errors.

In illustrative embodiments of the present invention, studies are performed at 1100 micromolar concentration of the meter. However, other concentrations may be used. The concentration may depend, for example, on the solubility of the meter and / or various toxins or other effects associated with the meter. In many cases, the concentrations used will depend on the sensitivity of the study.

- 17 010258

The purity of the target may be important or may not be important, for example, depending on the affinity of the meter in the presence of impurities and / or the sensitivity of the studies to impurities.

4. Illustrative studies

4.1 Functional research

Many types of functional research methods are known to those skilled in the art. In general, the treated target is provided with its natural substrate (for proteins) and the measurement of enzymatic activity is used to determine the functional effect of the meter relative to the baseline or controlled portion of the substance. Automated parallel research devices, such as those produced by Tesap (8 \\ 'Tskeg1apb). 2tag (USA) or Cyb (U) can perform multifunctional studies in parallel, for example, for different meters and / or for better statistics on a single correspondence meter-target.

Functional studies can be carried out at various levels, for example, at the molecular or cellular level of the body. In general, well-known functional studies can be used to investigate the functionality of a meter.

In an illustrative embodiment of the present invention, the meter acts like a target ligand and competes or otherwise affects the functionality of the target. Such effects can be of various types, for example, the meter can bind at the site of the alleged binding of a normal substrate, but, in addition, block the substrate from binding, the meter can bind in a way that does not block the substrate, but could if the meter was larger (suitable for binding studies), and / or the meter may be more agnostic than antagonistic in its behavior, enhancing the affinity of the substrate target.

DNA targets can be investigated, for example, using replication methods (for example, to see if replication is inhibited or enhanced). Alternatively, DNA targets are examined by determining their interaction with the DNA element after test binding. Such DNA elements typically include a substrate to which a plurality of short DNA segments are attached in a known order, with segments selected for binding (e.g., specific and complementary) to the parts sought for the DNA sequence and / or corresponding sections of the non-linear DNA segment. It is expected that the type and / or relative frequencies of binding to various short DNA segments in a DNA region depends on the degree and / or location of binding of the meter to the DNA molecule. For example, a meter may block a portion of a DNA molecule from matching with a segment of a DNA element. In another example, the meter may stimulate a conformational change in the DNA molecule, a change in which will interfere with binding to one segment of the DNA element, but which may allow binding to a previously inappropriate segment of the DNA element.

4.2 Binding Studies

In a binding assay, the binding of the meter to the target was directly measured. However, it should be noted that the binding assay may be less indicative than the functional assay because the meter can communicate with the outside of the target region and not provide useful information about the target region. Additionally, the sensitivity of the binding assay may be lower since the sensitivity of the search for binding is usually lower and, as a rule, the level of binding is also quite low. However, in some cases, functional research cannot be applied, for example, if the meter interacts with the substrate, or if the meter’s function is not known, or it can be difficult or time-consuming, for example, if living cells are required for the study. Also, the meter can bind in the active region without binding affecting functionality, as measured by a specific functional study.

Various types of binding assays known in the art can be used, for example, as described in Nabobox o1 Aegis 8bpcbb, ebbb ba KatakgEkpa 8e111a1a apb Prgabka vηΐΐιί V. Retapbes, w Egidb apb 111e Rkagtaseibs1, bbb Eekkeg, 2001, the disclosure of which is incorporated herein by reference.

Both functional research and binding studies can be performed in many ways using modern techniques, which include robotic test execution, and emerging methods that represent flow analysis (for example, using DNA elements). It should be noted that 100,000 test systems have become available, which means that some embodiments of the present invention using screening of a library of meters can be fully completed in one step (day).

This is optionally used in order not to clean the meter supply system between target screening.

In some embodiments of the present invention, a binding assay (functional assay) involves modifying the meter, for example, attaching a fluorescent substance to the meter. Depending on the point of attachment, this may cause conformational changes in some meters and / or cause spatial inconsistencies. It is assumed that overlapping between meters can overcome this problem, at least

- 18 010258 least, in a significant number of cases.

In other embodiments of the present invention, the meters do not change or do not change on a material level. For example, for NMR binding studies or binding studies by X-ray crystallography, a change is not required. In studies based on radioactivity, radioactive isotopes can be used in meters. In an exemplary embodiment of the present invention, non-radioactive isotopes (half-spin isotopes) are used in the manufacture of meters to provide binding searches and / or better studies of NMR data. In such studies, unbound meters can be separated from targets, for example, using methods known in the art, for example, if the target is bonded to a surface, washing can remove unbound meters.

In some embodiments of the present invention, the binding of the meter does not have a functional effect on the target, which can be found or measured, for example, exposure to the oscillation frequency of a fluorescent tail attached to the meter or target. In an illustrative embodiment of the present invention, the meter binds to the target in a manner similar to that of the target ligand. Various techniques can be used, for example, known in the art (e.g., NMR, IR), to analyze the combined target / meter structures. Optionally, after searching for a binding meter or other substrate, a set of meters is used to measure the combined target / meter structures.

In some binding studies, a plurality of meters labeled in various ways can be simultaneously investigated and possibly differently, for example, by attaching different fluorescent markers to different meters used together and / or using different radioactive isotopes for different meters.

Optionally, a binding assay (and / or functional assay) may include changing various environmental parameters, such as temperature, pH, and / or other environmental variables, for example, to determine binding strength.

In an illustrative embodiment of the present invention, a binding assay is used to determine the baseline of the level of binding of the measured active regions of the outside of the target. In one example, the degree of binding of a particular meter to protein alpha-helices may be known from a target analog. However, total binding to the target includes binding to non-helical portions of the protein and / or regions of the target.

In an illustrative embodiment of the present invention, it is noted that a large number of hits is expected and / or overlap is provided between the meters. As a result, a lower amount and / or faster investigation is used, since noise caused by low levels of binding can be a lesser problem. In one example, the boundary results of two studies are combined based on the repetition of triangular measurements between the meters used in the studies.

5. Meters, general view

5.1 Illustrative meter

FIG. 4A is a schematic illustration of an example meter 400 according to an embodiment of the present invention.

The meter 400 comprises a chassis 402 and four chemical groups 406, 410, 414 and 422 attached to the chassis 402 via bonds 404, 408, 412 and 420, respectively. This is only an illustrative meter, since the properties of all such elements may vary, for example, as described below. More specifically, one or more types of groups, the number of groups, the type of connection, the distance between the group and the frame, the type of frame, and the connection with the frame can vary for different meters, sets of meters and / or embodiments of the present invention.

In an illustrative embodiment of the present invention, many groups interact to determine a unit of measure. In an illustrative embodiment of the present invention, the purpose of the meter is to find places of interaction that bind to groups that define the unit of measurement and the distance between the groups. The correspondence of the unit of measure to the meter molecule can be indicated by linking the meter. In an illustrative embodiment of the present invention, the basic unit of measure is a triangle (or other geometric shape) defined by a subset of all groups. As will be described below, the shape of the triangle has specific properties that make it suitable for some embodiments of the invention. In general, if a meter includes more than a certain number of groups per unit of measurement (for example, more than two groups for a linear unit of measurement, more than three groups for a triangle), more than one unit of measurement can be provided with one meter. Thus, in an illustrative embodiment of the present invention, it is shown that many different triangular units of measurement are defined in one meter. In some embodiments, and for some reason, meters may include only one unit, for example, meter 400 includes only one four-point unit, but four triangular units. Illustrative ways to determine which of the different

- 19 010258 possible units of measure are really related, described below.

The geometry of one triangle is shown by dashed lines 416, 418 and 420, which determine the distance between the pairs of groups of the meter 400. As noted above, in an illustrative embodiment of the present invention, the task of the meter is to determine the places of interaction that are associated with such groups (406, 410, 414) at distances defined by dashed lines 416, 418 and 420 (for example, sides of a triangle). Provided that the meter 400 included only groups 406, 410 and 414, then the binding of the meter 400 to the target 100 can then be used as an indicator that three sites of interaction, of the type suitable for binding to groups 406, 410 and 414, are approximately at appropriate distances determined by meter 400. Since meter 400 defines multiple triangles, the binding of meter 400 indicates that at least one of the triangles defined by the groups is connected.

FIG. 4B shows a meter 400 interacting with a target 100 at three interaction sites 450, 452, and 454. Non-interacting groups and the remainder of the meter are not shown.

5.2 Number of groups per unit of measure

As noted, each set of groups defines a unit of measure. Although the present invention is oriented in some embodiments to two, three, four and / or another number of groups and / or sets of meters comprising a mixture of different units, in an illustrative embodiment of the present invention, the main unit of measure is a triangle with three groups. Using a triangle can provide one or more of the following potential benefits:

(a) A triangle defines a stable spatial relationship that can be suitable as a modular component when constructing a model in three-dimensional measurements of the target region, from the binding results.

(b) There are fewer possible triangles than four external units (for example). Thus, the creation of a library that includes units of measure that cover the entire space, less time consuming. In addition, as described in some embodiments of the present invention to provide overlap between units, such overlapping units are more easily provided if fewer units exist. It is possible that chemical constraints may not allow the design of meter libraries with higher-order units of measure.

(c) A triangle always lies in a plane (for example, three points define space), which may be mathematically suitable for some reconstruction methods.

(6) For some applications, the triangle represents the smallest number of binding points that can result in measurable binding to the active region of the target. A typical drug substance includes six or more binding points, often up to ten or more. In contrast, a higher order unit of measure may bind too tightly. In other applications, the optimal number of groups per unit, of course, may be higher or lower.

Alternatively, a unit of measure is used comprising two groups, for example, defining a line. Alternatively or additionally, a four or higher unit valency is used, for example, to more clearly determine the binding site configuration. In some embodiments of the present invention, a mixture of units of different valencies in a set of meters and / or during reconstruction, for example, 2-point, 3-point, 4-point and 5-point units of measurement, which may or may not be a plane, can be used. .

5.3 Number of groups in the meter

In an illustrative embodiment of the present invention, there is a certain number of groups in the meter, between four and ten, however, fewer (eg, three) or more may be provided. Some frameworks may be limited in number of different groups, group positions and / or possible group combinations. More groups are usually desirable if the groups define different triangular units. Conversely, meters with multiple attachment points and / or meters with many groups may be more susceptible to spatial mismatches and / or other adverse interactions between groups that inhibit binding.

Since the framework itself has chemical properties and can be considered as having groups, in some embodiments of the present invention, such properties are not taken into account, for example, during library development and / or during analysis of binding results. Alternatively, the properties of the framework can be taken into account, for example, only during analysis and / or during library development.

5.4 Group types

In an illustrative embodiment of the present invention, the groups are selected so that the types of bonds that the drug is believed to form with the target are reflected. In an illustrative embodiment of the present invention, groups are selected based on their

- 20 010258 chemical behavior. If a particular behavior is demonstrated by several groups, in the illustrative embodiment of the present invention, only one smallest of the groups is selected. In some embodiments of the present invention, multi-purpose groups that can bind to several different binding sites are used instead of groups that can bind to only one type of target site. The specificity of the selected group may depend, for example, on the total number of groups, their size and their susceptibility to chemical treatment. It should be noted that some groups are directional, while others are non-directional. Moreover, it is possible that undirected communications may be preferable to directed communications. In some illustrative embodiments, the implementation of the present invention perform two levels of measurement, a low resolution and a high resolution. More specific groups can be used in high resolution measurements. Additional details and methods for optionally reducing the number of groups used in some embodiments of the present invention are described below.

The following is a list of groups, one or more of which can be attached to the target:

a. Hydrogen bond donor. Directional communication.

b. Hydrogen bond acceptor. Directional communication.

c. Positive charge. Omnidirectional communication.

b. Negative charge. Omnidirectional communication.

e. Aromatic ring. Directional communication.

£. Hydrophobic group. Usually non-directional, however, sometimes, for example, a ring, can be directed preferably in the perpendicular direction to the plane of the ring. In other embodiments, different groups may also be used, for example, providing one or more halogen, carbonyl, phosphate and sulfate bonds. It should be noted that different groups can differ greatly in their chemical affinity or may differ less or even slightly. In some illustrative meter sets, a non-significant difference between group affinities is used to fine-tune measurement differences between types of relationships.

With respect to directional bonds, in some embodiments of the present invention, it is assumed that the bond has sufficient spatial flexibility so that a small number, for example, seven, of different directional bonds could be sufficient to cover all possible directions of bonds. Alternatively, fewer or more communication directions may be used. Optional different directional connections have a different number of directions presented in the library of meters. The angular distribution of directions can be, for example, uniform, or it can be uneven, for example, depending on the type of connection.

There may be several different sizes of hydrophobic bonds. In an illustrative embodiment of the present invention, two sizes are selected and provided in different groups. The aromatic ring may also serve as a large hydrophobic group. Alternatively or additionally, an aromatic ring is used to match aromatic bonds with other rings and / or certain types of hydrogen bonds.

In the above selection of groups and directions, 25 special groups are obtained that can be attached to the chassis. An illustrative set of groups is described below.

In an illustrative embodiment of the present invention, a subset of the above groups is used. The rotational flexibility property of the hydrogen bond donors and / or their recipients is used. Although this flexibility usually reduces the likelihood of chemical bonding, the mass of the hydrogen atom used in the hydrogen-bonded group is low enough so that a decrease in the probability may not significantly affect the results of the measurement method, at least for some meters and studies.

Alternatively or additionally, rotational flexibility is acceptable for aromatic rings. Although aromatic rings have a high mass, the large bond area of the ring compensates for the decrease in binding strength caused by the possible rotational flexibility of the ring.

Alternatively or additionally, some polar bonds can be represented by one group, such as OH, which can act as a hydrogen bond donor and an acceptor.

Optionally, for example, if chemical information can be dispensed with, more conventional groups are used and fewer triangles in the library are covered.

5.5 Overlapping units in a set

In an illustrative embodiment of the present invention, the space of triangles, as a whole, is encompassed by providing a plurality of triangles, each with sufficient freedom in its parameters (e.g., bond length, chemical affinity), so that it is assumed that each triangular arrangement of the binding points is associated with one of the triangles with a measured degree. Optionally, the coverage of each triangle in the space of the triangles is covered by the coating of other triangles to ensure that there are no uncovered parts

- 21 010258 spaces.

As will be explained in more detail below, in an illustrative embodiment of the present invention, the gauge library is designed so that each possible triangular arrangement of the binding points is found (or matched in parameters) by more than one gauge. In some cases, completely congruent triangles cannot be provided, except for providing triangles that are approximately congruent (for example, similar groups, lengths of sides). Such congruent triangles may or may not have the same coverage in the space of triangles. For example, provided the same groups are provided, two triangles with the following lengths of the sides: (3, 4, 5) and (3.1; 3.9; 5.2) (measurements in angstroms). Such triangles can, for example, cover part of the space of triangles from (2, 3, 4) or (4, 5, 6).

In some embodiments, implementation of the present invention, at least some space of the triangles covered by a set of triangles with overlapping coating. For example, for the same part of the space of triangles, the provided triangles are (2, 3, 4,5) and (2,5; 3,5; 5,3), which have overlapping but different coverage.

Since overlapping is suitable for different occasions, for example, as described below, it really increases the size of the library. If overlap is provided, the reconstruction method used does not necessarily take into account the overlap.

6. Reconstruction

After the method 300 (Fig. 3) is repeated for the required number of meters, the measured affinity of the meter 400 to the target 100 is optionally used to reconstruct the spatial distribution model of the interaction region 102. An illustrative method is described below.

In an illustrative (theoretical) method for mapping a specific target molecule that uses a library with 75,000 meters, it is estimated that approximately 400 meters can communicate with the target. Due to repetition of triangles in the library and / or due to overlapping coverage of incongruent triangles in the illustrative library, the number of real triangles defined by the target area and connected by meters is assumed to be less. In one (theoretical) example, the number of real triangles, which is determined by the target area and connected by meters, is 100 different triangles.

Taking as an example a 10-point pharmacophore, such a pharmacophore may include, for example, 10 * 9 * 8/6 triangles, which is 120 triangles. In some embodiments, the implementation of the present invention, not all such triangles are identified, for example, due to the high similarity between the triangles (below the possibility of discrimination) or due to the lack of binding (for example, due to spatial inconsistencies). Of course, a 10-point structure can be reconstructed with less than 100% triangles, especially if the missing triangles are missing randomly. For example, 50% of the triangles may be sufficient.

However, in a real situation, the requirements are lower. A typical pharmacophore may include 20 points, of which, to ensure good binding, usually only 8 to 10 points must be identified. Thus, any pharmacophore structure that includes 8-10 correct points can serve as a good starting point for the production of a drug substance. A smaller number of identified points may also be suitable, for example, as described below.

Although various methods may be used to reconstruct the arrangement, a two-stage method is used in an illustrative embodiment of the present invention. First, real triangles are evaluated from the results of the study, optionally using a clustering algorithm. A suitable arrangement using triangles is then found, optionally using an evaluation-based search algorithm or a clustering algorithm. In other embodiments, one-step or multi-step methods can be used.

6.1 Selection of triangles

In an illustrative embodiment of the present invention, this method step has two parts, however, in other embodiments, this step has one part or more than two parts. In one part, it is determined which units of the triangles correspond. This part may be less trivial, for example, due to the fact that each meter includes a large number of triangles. However, repeating triangles between meters can help with discrimination. Another optional part of the method is to determine the actual included distances, and not those determined by the unit of measurement. For example, the real distance between the two groups can be 4.3 A, while the units of the binding triangles have distances of 4 and 5 A. In some embodiments of the present invention, it is desirable to estimate the real distance of 4.3 A from the results of the binding. Optionally, this is achieved by overlapping the coverage of different units of the triangles.

In an illustrative embodiment of the present invention, two parts of the method are provided in one complex method, for example, using clustering.

Alternatively, a two-step process can be used. Iterator optional

- 22 010258 method with an assessment of which units are connected, which are used to estimate real distances, and real distances are used to improve an earlier estimate of which units are connected.

FIG. 5 is a flowchart of a method 500 for determining which triangles actually associate with a target according to an illustrative embodiment of the present invention.

At 502, a space is determined for each type of triangle (defined using triangle groups). Each such space has three dimensions, each dimension representing the length of the side of the triangle.

At step 504, a space is marked in the place {x, beats} if it is shown that a meter including this type of triangle with side lengths {x, beats} is associated with the target. It should be noted that for two different frames, the exact correspondence of triangles can be difficult to create. In addition, the triangles can be almost appropriate, for example, having an insignificant difference in the lengths of the sides.

In an illustrative embodiment of the present invention, the results of the study are used as binary input to determine whether the relationship is lighter or not. Alternatively, for example, if conformational changes are observed or a unit for measuring activity and / or binding exists, binding strength can be provided using continuous or multi-stage amplitudes using hit marks.

In an illustrative embodiment of the present invention, if one meter includes a large number of triangles, a hit is noted in each one of the relevant spaces. Alternatively or additionally, if one triangle can correspond to two different types of triangles, for example, due to overlap between the affinity of groups, it is also noted in a large number of spaces. Optionally, the amplitude of the mark is normalized to the number of spaces that is marked by the meter.

Alternatively or additionally, in each space different amplitudes are provided that are sensitive to the a priori probability of binding.

In step 506, hit marks are restored by the spatial distribution function. In an illustrative embodiment of the present invention, the propagation function is the probability that the triangle forms a bond at different distances represented by coverage. Alternatively or additionally, the coverage is between spaces, for example, if two groups overlap with their affinity.

Alternatively, a hit indicator is initially provided as a distribution function.

In an illustrative embodiment of the present invention, coverage is defined as

where Dx is the difference between the lengths of the sides, and ah is the value representing the difficulty of binding the molecule so that it can form a bond. In an illustrative embodiment of the present invention, σ x is a function of x, for example, σ x = a7x. In an illustrative embodiment of the present invention, parameter a is 1.414. Perhaps the propagation function is heterogeneous in space, for example, to display the characteristics of the heterogeneity of the connection. Optionally, at least some of the propagation functions are derived empirically using binding meters having controllable distances between bonds with targets having known models. Alternatively or additionally, such empirical testing is used for other purposes, for example, to determine flexibility in bond lengths, multiple chemical affinity of groups, and / or symmetry of the propagation function. Optionally, targets are also classified according to their flexibility. Optionally, in the iterative process, after evaluating the model, the flexibility of the target is evaluated and / or they decide, for example, to form a table and use to adjust the propagation function used.

Then the covered hits are combined, for example, by adding, and as a result (508) peaks are found. In an illustrative embodiment of the present invention, peaks are selected based on their shape. Alternatively or additionally, the peaks are selected based on their amplitude crossing the threshold. Such a threshold may be, for example, the number of triangles that is necessary for communication, to indicate a possible match. The threshold can be the same dx: I am of all spaces or it can be different. Optionally, a threshold and / or decision method is selected based on clustering statistics, for example, from a table of previous empirical results. Alternatively or additionally, the threshold is chosen such that a minimum number of matches are found. Optionally, if there are a large number of sub-threshold matches, a different set of meters is used for the binding process. It is noted that in some embodiments of the present invention, for any given triplet of attachment points, typically there are 12 triangles or more that are thought to be capable of binding. For example,

- 23 010258 it is assumed that both the shorter side and the longer side are associated with a pair of binding sites having an intermediate distance between them. Additionally, each type of triangle can appear repeatedly, for example, three times in a set. In some sets, each (or some) point of a triangle in the space of triangles is covered by 24 triangles - 8 triangles are designed so that they have short and long sides in various combinations, 3 times, if each triangle is provided 3 times. Additional overlapping can be achieved using ambiguous groups.

Optionally, using a correlation analysis between spaces and meters, a four-point (or more) geometric match can also be found.

6.2 Reconstructing a configuration layout

FIG. 6A is a flowchart of a method 600 for determining the spatial arrangement of binding sites from the results of the method of FIG. 5 according to an illustrative embodiment of the present invention. In an illustrative embodiment of the present invention, the method comprises constructing all the configurations (e.g., three-dimensional shapes) that can be constructed from the identified triangles and ranking the configurations using the rating method, ultimately selecting the configuration with the highest rating.

In step 602, all possible configurations that can be constructed from the triangles found in FIG. 5. Alternatively, to create a computational model of all possible configurations in an illustrative embodiment of the present invention, configurations are specially created. For example, in combination with the evaluation method described above, a configuration can be designed or its design improved only if it is suitable to have a suitable evaluation. For example, as soon as a configuration solution has a rating lower than the largest one found, such a lower solution is ignored.

In an illustrative embodiment of the present invention, the construction method is created by gradually building the structure fragment by fragment. For example, a triangle is added to an existing configuration only if there is a side and / or group length corresponding to the side length with a pair of groups on the structure. The size difference threshold can be determined to allow two sides to fit. Alternatively or additionally, the limit of correspondence between groups can be determined. Optionally, groups are required for matching at the ends of the corresponding side or having overlapping chemical behavior. Such thresholds may depend on the length and / or types of groups and / or other properties of the meters and / or target. It is noted that the first meter can bind to a specific binding site using a binding method different from the second meter, provided that this binding site supports both binding methods.

In an illustrative embodiment of the present invention, the configuration design is the sequential selection of a triangle from the list of available (connected) triangles until all the triangles are fully used. Used triangles may remain on the list for reuse. Alternatively, the configuration can be gradually built using modules, each of which is constructed from submodels and, ultimately, from triangles.

At 604, an estimate is calculated for each configuration. Such an assessment does not necessarily represent a heuristic value, indicating the validity of the research results derived from targets with a configuration. Various methods of calculating the score may be used. In an illustrative embodiment of the present invention, the method of calculating the estimate is based on a specific joint connection of the triangles in the configuration and / or on the possibility of the triangles themselves to be initially true.

In an illustrative embodiment of the present invention, the score is the product of the calculation of the score for each common side of the triangle. In an illustrative embodiment of the present invention, an estimate for the side of a triangle that is common between two triangles is the estimated probability of two sides of two triangles linking to the same pair of binding sites. In an illustrative embodiment of the present invention, the estimate is the product of the above propagation function for the x, y, and ζ axes. Alternatively or additionally, other, simpler estimates can be used, for example, based only on the difference in the sizes of the sides.

In an illustrative embodiment of the present invention, the score is independent of the lack of triangles. For example, if the created configuration includes a three-point configuration for which there is no suitable correspondence between the meters, it is not assumed that the configuration is incorrect, but the estimate is underestimated. Alternatively, the estimate may be underestimated in response to the existence of triangles that are found in the configuration and not found on any appropriate meter, for example, based on their calculation.

Alternatively or additionally, some configurations can be controlled based on heuristics, for example, rules that describe which layout usually looks appropriate.

- 24 010258

Alternatively or additionally, previous information is used to control certain configurations, for example, an incomplete model or knowledge of a molecule that binds well to a target.

At 606, the highest-rated structure is selected as the map of the layout of the binding sites of the target. As noted above, steps 602-606 can be performed as an iterative search and construction method, for example, with structures that are specially built in the process of advancing the search, and indicates certain structures that have an estimate above the threshold (and therefore may depend on it). Many suitable search methods are known in the art, for example, in the field of graph search and in the field of a game search tree (for example, for chess game programs).

6.3 Reconstruction options

In an illustrative embodiment, the target may have several active regions. In an illustrative embodiment, the reconstruction allows you to re-create the structure of non-overlapping configurations, with each non-overlapping part of the map of one area of the target. Optionally, such a reconstruction may be necessary even for one active region, if there are enough triangles (for example, groups of gauges) that connect unconnected parts, are unsuitable for binding (for various reasons) and / or were not available in the used set of gauges in such a way that a continuous structure cannot be reconstructed from triangles that really match.

Optionally, the above reconstruction allows the triangle to appear only once in the reconstructed configuration. Even if a triangle does appear twice (or more) in the actual configuration, redundancy of such triangles can usually still be possible to reconstruct the structure. Alternatively or additionally, it is permissible for a triangle to appear more than once, however, this may affect the estimate, for example, reducing it. Alternatively, an iterative experimental approach is used as described above to block part of the target (for example, with a suitable antibody or small molecule drug substance) and observe if the triangle still matches.

Optional user intervention is possible, for example, to consider the final structure or some candidate structures. For example, if the calculation cannot be done, a person can make a choice among the options, strengthen the influence of some correspondences and / or parts of the configuration and / or remove some options from the analysis based on, for example, human experience and discretion and / or additional information about the target of various types.

It should be noted that the possible result of clustering and / or the method of reconstructing the form is the input data for the interactive process and / or further development of the drug substance. For example, the application of the above methods can show where there is not enough more accurate data to create a complete result and / or where there are ambiguities between possible solutions.

It should be noted that the resulting structure may have a mirror (for example, symmetry) ambiguity due to the use of only triangles. Optionally, such ambiguity is resolved by using at least one 4 or more point unit of measurement, optionally designed or selected for linking in only one embodiment. Alternatively or additionally, the effect of spatial inconsistencies is used to distinguish between options. Alternatively or additionally, the previous information is used to distinguish them.

6.4 Alternative reconstruction methods

FIG. 6B is a flowchart of an alternative reconstruction method 620 using clustering of shape reconstruction according to an illustrative embodiment of the present invention.

In step 622, a triangle is selected from the set of found triangles that were found for linking in the study and clustering of FIG. 5. Such a triangle is used as the basis for constructing the structure.

In step 624, a pair of triangles is selected from the remaining triangles found so that two triangles share a side with each other, and each triangle also shares a side with a part of the structure (for example, in which two sides of the structure could or could not be sides of the same triangle, depending, for example, on the implementation). When a pair of triangles is added to the structure, the structure grows by one point in space.

Step 624 is repeated (626) until a pair of triangles can be added. This complements one potential structure.

Often there are several possible options in step 624, for example, to select pairs of triangles, and / or search where to add them. At step 628, three possible structures are performed by repeating steps 624 and 626 for each possible variant of the pairs of triangles and their place. This process can be performed, a priori, for example, by exhaustive search of multiple threads each time when multiple pairs of triangles are available for selection and / or each time such pairs can be attached to different places.

- 25 010258

At step 630, steps 622-628 are repeated by selecting all possible triangles as the base triangles, in turn (or in parallel). Alternatively, other methods of creating all possible structures from triangles can be used. Optionally, a drop method is used, for example, if the structure is clearly unsuitable or impossible to use a significant percentage of triangles (for example, 30, 50, 70% or any smaller, medium or larger value), the structure is excluded. Usually, the more triangles you can ignore, the easier it is to get a structure (for example, even in noise). However, the structure may be less limited by research results and may be less dependent.

At step 632, all structures are found in the created potential structure. Optionally, only some substructures are found, for example, only the largest or only those that are larger than a certain size. In an illustrative embodiment of the present invention, the method used is the most likely algorithm for finding the most likely structure.

At step 634, such substructures are clustered, with each point representing a structure in which the substructure is found. In an illustrative embodiment of the present invention, the clustering space is determined for the type of triangle (for example, the type of substituent in the triangle), and the space is enclosed by the sides of the triangles. Thus, for example, a 10-point substructure of a 20-point structure is created in a space that includes the same number of types of groups as a substrate with a place in space defined by three Cartesian locations of each of the points (for example, 30 measurements for a 10-point space) . For various orientations, they do not necessarily deal with the choice of the main triangle as the base triangle. Alternatively or additionally, space is created by structures in a rotationally symmetrical manner (or thus analyzed) so that results from different orientations are comparable. An illustrative algorithm is described by J. Νιΐδδίηον. NE. HUoizop. in EGysuy Osuyeyop oi Tgee Oipep5yupa1 81gis1iga1 MOG ίη Vyuofsa1 Masgoto1esi1e8 уu SotriYug Ukyup Tesyshdiez, ΡΝΑ8, Volume 88, pp. 10495-10499, December 1991, incorporated herein by reference.

At step 636, the best structure is selected. It is assumed that if the substructure is sufficiently general and large enough, then it is both correct and suitable. In an illustrative embodiment of the present invention, a threshold is used to select only those substructures with structures and clusters that are above a minimum value. Other selection methods may also be applicable, for example, coverage, for example, based on an accumulated assessment of the correspondence of pairs of triangles (such a correspondence can be threshold during construction, for example, using a given threshold).

Alternatively, other methods are used to search for large common substructures.

It should be noted that since the clustering method can create structures that do not really use all the triangles and are incomplete, a complete pharmacophore map is not essential for many embodiments of the present invention, for example, to create and search for a leader.

7. Analysis

7.1 Overview

The above method of measuring and reconstructing a target region can be used to provide a wide range of information. The quality of information and its type can be of different types. The following are illustrative types of parameters that can be used to classify such information:

(a) Completeness. Information can be full or partial, for example, a complete model of the target area or a model of only part of the area.

(b) Factual or statistical. An example of factual information is an accurate model. An example of statistical information is a set of relative options for a set of probable models.

(c) Independence. The information may be independent of other information, for example, be an exact model, or it may be dependent, for example, a parametric model, whose exact value depends on additional information. Additionally, information derived using the above methods can be used as part of the information of various processes.

(6) Justification. Information may be supported by other information, or it may exist on its own, or even conflict with other information.

(e) Positivity. Information can be positive in that it indicates that it exists, if desired, or in negative, so that it can be used mainly to remove some possibilities.

Since the information collected may be about binding sites, in one case, the information also relates to the geometry of the target not at the binding sites. As will be described below, for example, the geometric structure may also affect the suitability of the drug leader.

In some embodiments of the present invention, analysis is used to obtain

- 26 010258 information about the targets themselves, for example, their relative binding affinity and / or their chemical behavior (for example, pH dependence). Such information may be ordinary, or it may be for target groups, for example, different for different families of proteins and the same within the family.

As can be taken into account, such a widely varying range of information may be subject to many analysis methods, some of which are described below, and for many applications, some of which are also described below. More specifically, some illustrative methods of analysis are aimed at creating additional information about the target area and for determining errors and analysis, and some illustrative applications are combined as part of a method for searching for a drug substance.

In some cases, the results of the analysis are combined during reconstruction as geometric and / or chemical information. Alternatively or additionally, the information is related to the reconstruction and / or target, for example, in a manner similar to the method used for the drug leader. This method usually depends on the type of database used to store the information.

7.2 Verification of reconstruction

In an illustrative embodiment of the present invention, the size and / or type of layout error is determined. In one embodiment, the reconstructed arrangement is analyzed to create theoretical binding values for the set of meters used. Differences between such theoretical binding values and actual binding values can be used to indicate parts of the layout that are not accurate and / or to indicate the degree of inaccuracy of the layout and / or the entire reconstruction process.

Alternatively or additionally, physical verification is used, for example, by applying an additional testing method and / or research library to choose between alternatives or for verification.

7.3 Binding Strength

In an illustrative embodiment of the present invention, the created arrangement is analyzed to evaluate the relative binding strength of the binding points in the target region. In an illustrative embodiment of the present invention, the reconstructed arrangement is simulated and theoretical binding values for a set of meters are calculated. The difference in binding values may be partially due to a decrease or increase in the affinity of the target region. Such estimates are usually statistical in nature, since there are a large number of variables that affect the likelihood of binding. However, it is assumed that if the length and type of bond are known and the exact positioning of the meter in the target region (for example, and its energy sequences) can be determined, then at least a statistical analysis of the binding strength can be provided. Optionally, by analyzing molecules with known behavior or by comparing the binding of different but similar measuring triangles, a baseline is provided.

7.4 Interactions within relationships

In an illustrative embodiment of the present invention, an assay is used to determine the interaction between the binding of different binding points. For example, such an analysis can compare the contribution of the binding point to the binding of some meter compared to what is expected (for example, based on energy and other calculations) and / or compared to the observed contribution of such a binding point when binding to another meter. This may indicate, for example, the effect of binding at one site of interaction on the affinity of neighboring sites of interaction. Optionally, such interactions are evaluated and / or modeled using a model of electric charge distribution in the target.

7.5 Geometric analysis

For some tasks and to some degree of accuracy, it is believed that a particular layout may be a cast of the target area. In an illustrative embodiment of the present invention, the geometry of the target region is analyzed. Additional information can be provided by determining which meters did not bind or bind with a lower affinity (which is assumed that, if the binding geometry was similar in some embodiments of the present invention, is due to spatial inconsistencies). This can help in further determining the geometry of the target region. It should be noted that some spatial mismatches can be predicted from the layout geometry. For any unsuccessful binding, which has no other obvious reasons and must correspond to a certain geometry, it can be assumed that this is the result of protruding matter that does not define a noticeable binding point. This is described in more detail below.

In an illustrative embodiment of the present invention, geometric analysis is used to determine the size of the inlet in region 104 (for example, where arrow 400 is shown in FIG. 4B). A small hole and / or some groups at the entrance of the hole may preclude the possibility of certain sizes and / or types of drug substance. Alternative or Complementary

- 27 010258 but, geometric analysis is used to classify the target, for example, based on the size of the substrate on which it can work. In an illustrative embodiment of the present invention, geometric analysis (for example, to determine the substrate) is supported by chemical analysis of groups of the target region 104. Geometry can also be useful in deciding which methods of labeling small molecules and / or meters can work (for example, without using large fluorescent markers if the inlet is small).

It should be assumed that in some cases it may be easier to reconstruct the geometry of the target region than the pattern of its chemical binding or vice versa.

7.6 Determination of spatial discrepancies

In an illustrative embodiment of the present invention, spatial discrepancies are determined in the analysis method and / or used to provide additional geometric and / or chemical information about the target. In an illustrative embodiment of the present invention, spatial inconsistencies during the binding process are determined by comparing the affinities of different meters with the same triangles. Such a comparison does not necessarily take into account one or more inlets, the chemical behavior of the meter, the degree of conformity to the binding geometry and / or other binding sites. Spatial mismatches, for example, arise when close or potential overlapping of the meter and the target molecule reduces the binding affinity.

Since the shape of the meter is known, in some embodiments of the present invention, it can be assumed that relatively rigid spatial mismatches are the result of non-participating groups of the meter and / or the frame itself.

In an illustrative embodiment of the present invention, spatial mismatches are used to create a map of places near the target that interact with the atoms of the meter, thus possibly indicating occupied (for example, atoms, electric fields) parts of the target that obviously do not cause binding interaction with any meter to a noticeable extent.

In an illustrative embodiment of the present invention, the map is used to provide additional information about the shape of the active region in the target 100. Alternatively or additionally, the map is used to help develop a drug substance, for example, by filtering out potential drug substances that might have the same spatial inconsistencies . Optionally, a certain level of filtration can be achieved by simply matching the geometry of the drug substance to the geometry of the meters, which should have a good binding but no binding.

An analysis of geometric and / or chemical affinity can also be used to determine the shape of the natural substrate of the target, for example, if it is clearly unknown, and / or to determine how much of the substrate is occupied by the target 104.

7.7 Identification of control areas

In an illustrative embodiment of the present invention, the results of binding and / or reconstruction are analyzed to find one or more control regions of the target. Typically, control regions are not associated with the main substrate of the target, instead of binding to a single hormone or other modified molecule. Such secondary binding usually affects the binding behavior of the target region.

In an illustrative embodiment of the present invention, the control areas are determined by the size and detachment from the reconstruction of the layout of the main area of the target. Alternatively or additionally, the control regions are identified by test bindings to pairs of meters (either in the presence of various molecules, optionally selected a priori or after determining the presence of control regions) to search for the binding relationship within the meter. Alternatively or additionally, control regions are identified based on the shape of the reconstructed layout. Alternatively or additionally, the presence of control regions is found by the presence of the remaining bindings of the meters, which are not necessary or not installed during the reconstruction.

In an illustrative embodiment of the present invention, depending on whether or not binding to a control region is desired, various identifications of control regions can be used to screen for drug leaders.

7.8 Other types of map analysis

A map or model of the target can be analyzed to collect other information according to exemplary embodiments of the present invention. For example, as noted above, the distance of the binding point from the control region or active region can affect the type of drug search. For example, a drug substance that binds to a control region may have an enhancing effect on the target, for example, an agonist. A molecule that binds near the control region or the active region, or inside the active region, can affect the target so that it becomes less sensitive to signals and / or incapable

- 28 010258 to act, for example, an antagonistic effect. Thus, in an illustrative embodiment of the present invention, the site of the binding region on the target is used to help determine what type of therapeutic effect is expected from the developed drug substance. For example, the binding region near the region of the target may indicate a drug substance whose tail blocks access to the region of the target.

In another example, the binding regions that are located on the outside of the target region can be used to improve drug development.

A drug substance can be designed (or found) to include parts that bind to the target region and parts that bind to the outside of the target region. The combination of binding regions provides a binding force greater than that provided separately by each region, since the part of the molecule associated with the region of the target can provide the desired therapeutic effect. Alternatively or additionally, a molecule that binds to two separate regions can cause conformational changes or prevent such changes in the target molecule.

8. The use in methods of searching for a medicinal substance

8.1 Overview

The search for a drug substance is a very long and expensive process by which drugs are found to treat diseases. The process begins by identifying a target that is exposed to a drug substance, searching for potential drug substances that act on the target, and then determining which, if any, of the potential drug substances is safe and reliable. Often a suitable drug substance is not found and one of the drug candidates is modified in various ways in an attempt to make it more suitable. One reason for the complexity of the drug search process is the difficulty in determining which molecule can act on the target. As will be described below, in some embodiments of the present invention, the methods of the present invention are used, at least to reduce to some extent such difficulties. Another reason for the difficulty lies in the many unexpected effects of potential drug substances that make them unsuitable and / or predictable. On the contrary, as described below, some of the methods of the present invention can be used, at least to reduce to some extent such difficulties.

Usually, by the methods of searching for medicinal substances, they try to answer two questions. The first is whether there is / which molecule of the drug substance exists, which strongly binds and acts on the target molecule. The second is how to evaluate that such drug molecules have a ΆΌΜΕΤ properties profile (ΆΌΜΕΤ means Toxicity of the Absorption Distribution of metabolic Excretion), which ensures success in clinical trials. In an illustrative embodiment of the present invention, the method, materials and / or devices described herein are used to select, develop and / or determine a goal directed towards potential drug molecules that have chemical characteristics known or suggesting an improvement in profile. Lipinsky's rules are an example. It should be taken into account that, knowing which parts of the molecule are important for binding and which are not (in accordance, for example, comparing the molecule with the target model), one can easily modify (or plan for improvement) potential compounds of drug leaders for strong binding and / or satisfying any well-defined qualities.

In the general case, the above methods and especially various target models may be useful in selecting molecules or searching for methods that match the map and reject those that do not match. Other methods of the above measurement processes are also described below, for example, using an additional mapping step to test the theory. As can be assumed, various methods (existing and new) for the search for drug substances can be used in different ways by applying illustrative embodiments of the present invention.

It will be appreciated that various embodiments of the present invention may be performed in an automated manner. However, due to costly analyzes in some embodiments of the present invention, the application is semi-automatic, for example, using methods to change the search process, for example, by adding a filtering step or a candidate creation step, since it is still possible to use a person’s opinion, for example, a solution, are whether some generalized and vague criteria are appropriate. In some cases, the entire process is controlled using human intelligence along with one of the various steps, for example, manual mapping and / or rejection of leaders. For example, changing thresholds and re-executing a step is, for example, a decision that can be left to a person.

8.2 Creating a drug

One relatively new type of drug discovery is actually the creation of

- 29 010258 of a new drug substance, the development of a new molecule in order to have the desired functions. In an illustrative embodiment of the present invention, the above chemical and / or geometric map of the target is used to assist in this process. For example, the synthesis may be accompanied by a demonstration of what form the active part of the drug substance should have (or limit the range of possible forms).

In an illustrative embodiment of the present invention, the synthesis of a drug substance involves obtaining meters from a library and modifying them, for example, replacing groups so that they better match the target. In some cases, the replaced groups have the same affinity, but different binding strength, for example, when choosing ΝΗ ₂ or OH for the hydrogen bond donor, and choosing the optimal size of the hydrophobic group. It should be borne in mind that a force based on the classification of groups can also be used in the construction of the library, for example, by providing numerous forces of hydrogen donors or numerous sizes or hydrophobic groups. One possible application is to achieve a better uniformity of bonding strength between groups. Another is to provide libraries with higher precision.

In an illustrative embodiment of the present invention, scaffolds and / or parts of molecules for use in the synthesis of drugs are constructed as a result of target mapping processes. For example, by analyzing the geometry of the target, a set of frames can be found that covers (with attached groups) the largest target spaces. Specific targets can, for example, be constructed from subframes or selected from libraries of chemicals, for example, using a method similar to that described below, to search for meters in libraries. Group sets or group clusters can be selected, for example, based on statistical analysis, which groups are clustered together in the target family or in the general list of targets. Optionally, targets are selected to be illustrative of prospective future targets. Of course, it is assumed that in some cases an ideal drug substance cannot be created using such synthesis methods, but a synthesized drug substance may be a good starting point for improving the drug substance.

8.3 Creating a Leader

Often, it is easier than the synthesis of a drug substance to create a leader in which a leader that is not expected to be a suitable drug substance is created and then improved and modified using methods known in the art. In an illustrative embodiment of the present invention, a map is used to describe a potential molecule for synthesis as a drug leader. In an illustrative embodiment of the present invention, a map is used as a set of constraints, and a search is made to search for a molecule that matches the constraints. Additional limitations may be, for example, known synthetic methods, the shape of the base molecule used as a starting point. Illustrative software that may be used is IPN1 sold by IP8 (USA). The bcc system works by attaching basic chemical components in order to obtain the desired correspondence — a pharmacophore or other molecule.

Then a potential molecule can be synthesized and improved to a drug substance, as is well known to specialists in this field of technology.

In an alternative method, a potential drug molecule can be constructed by combining molecules of a library of meters or other molecules having suitable groups or structures together so that the resulting molecule has a higher affinity than a single meter. Such a molecule can then be optimized, for example, by removing unnecessary groups and / or joining groups to provide various desired properties. Optionally, the meters are connected using a chassis, and not directly to each other. Optionally, by analyzing which meters are connected (for example, using clustering), it is possible to achieve a better estimate of the desired size and / or chemical properties of the fragments to be joined. For example, the choice of two meters in order to connect them together can be based on the actual binding of additional (or other meters), for example, 2, 4, 5, 6 or more meters. For each such set of meters that binds, the best meter or other molecules are selected for the connection. Alternatively or additionally, meters with more specificity are used to determine which of several possible measurements of the triangle of the meter is actually connected. Such meters with higher specificity can be created, for example, by removing groups from existing meters (or creating such meters using any method known in the art). Such meters with higher specificity can also be used for other embodiments of the present invention, for example, to improve clustering statistics. However, usually because of the relatively large number of possible such meters, they are used if there is a way to limit the range of possible triangles. Alternatively, a large number of meters with higher specificity, for example, meters with 1, 2, 3 or 4 triangles

- 30 010258 mi designed to be used as a library or part of a meter library.

In an illustrative embodiment of the present invention, knowledge of the target structure is used to accurately determine the location of the linker and / or to select a suitable linker that avoids spatial mismatches with the target.

In an illustrative embodiment of the present invention, the meters for the connection are actually selected without constructing a model. Instead, binding meters are selected and joined together. Alternatively, a model is used to decide which meters to connect and how to connect them. Such a model can also be used in other leader connection schemes, for example, as described in the prior art to guide the selection of which fragments to join to provide the length of the connection, where to attach and / or in which orientation to attach. Optionally, leaders are constructed synchronously from the meters and each step is checked to see if it matches the expected behavior.

Alternatively or additionally, instead of using a model as described herein, various types of models are used, for example, a target model associated with a meter, for example, created using x-ray crystallography and / or NMR. Such a model, for example, once created for each set of meters can be used to decide what distance and type of connection to provide to create a leader from the meters. Alternatively or additionally, a new molecule can be designed and constructed so as to have binding points at some or all of the locations shown by a crystallographic model for binding, for example, two, three or more meters. In the general case, this type of method assumes that after the configuration of the coupled target-meter pair becomes known, an exact model may not be necessary, since real matching information is available.

Alternatively or additionally, as noted herein, a measurement of a bound target can be made.

8.4 Leader Description

In an illustrative embodiment of the present invention, a map is used to describe one or more profiles of molecules that can be assumed to have an effect on the target. In an illustrative embodiment of the present invention, the created profiles take into account one or more of the following:

(a) the geometry of the layout of the interaction site;

(b) the affinity of the sites of interaction;

(c) the size of the entrance to the active region (s);

(6) identification of a potential control area (s);

(f) synthesis ability; and (D) extensibility, for example, that additional groups can be attached.

Typically, a molecule requires at least five or six bonds, strong enough adhesion to the target to act on the target in nanomolar concentrations. The exact amount may depend, for example, on the affinity of the sites of interaction. A single target can usually provide a large number of possible profiles. Such profiles may correspond to libraries, for example, using methods known in the art.

In an illustrative embodiment of the present invention, profiles are created using a format that corresponds to particular search software and / or library data structure. In an exemplary embodiment of the present invention, a pharmacophore search is provided, for example, as is known in the 1818 (MO) database (when searched in 3Ό databases).

8.5 Leader Search

In an illustrative embodiment of the present invention, a map is used to search a library of known molecules for possible matching. Perhaps the map is used instead of analytical target models in well-known virtual scanning techniques. In an illustrative embodiment of the present invention, the library is pre-processed so that the molecules in the library are described in terms of groups and model geometry of the layout and / or targets used in measuring the target. Alternatively or additionally, the existing library is pre-processed to collect descriptions of the compared meters from its contents, for example, each molecule is defined as a parametric model based on the meters for measuring. It should be noted that such a description may not be a one-to-one mapping, for example, the same molecule can be described using two different sets of groups, since there is some overlap between the chemical behavior of the groups.

In an illustrative embodiment of the present invention, potential leaders are identified based on their inclusion or the ability to include a large number of groups in the required positions, as indicated by map. In one embodiment, the search is performed for 3 points or more (for example, 4, 5, 6, 7 or more) matches. In another embodiment, each molecule in the library is checked for the number of groups that it includes at the required positions.

- 31 010258 and on the suitability of the connection points for joining the missing groups. In an illustrative embodiment of the present invention, the missing groups are added one after another until a suitable drug leader is created (e.g., binding of sufficient strength).

Illustrative search is performed by the base 1818 (ME).

One possible type of search involves taking into account all suitable 3Ό structures, and when searching, they are divided into sets and / or subsets of pharmacophore points and a search is performed to establish within the tolerance range defined in the request.

8.6 Leader rejection

In an illustrative embodiment of the present invention, the results of the above methods are used to reject leaders that otherwise look suitable. In one example, a leader (or family of leaders) is rejected if the above models imply a lack of binding and / or spatial inconsistencies. In another example, they make the assumption that if the leader is suitable, it is believed that the meter corresponding to the triangle (or other number) of groups on the leader is associated with the target. If such a meter is not found or data analysis implies that the likelihood of a triangle connecting three groups in the meter is unlikely to occur, leaders are rejected or investigated further. Alternatively or additionally, the correspondence of some measure may also indicate that the leader is not suitable.

In one example, those skilled in the art can use the information provided to determine if a leader is suitable for optimization (enough). For example, it is suggested that by directly adding or removing certain groups (for example, which are often considered the main types of small changes in a molecule), affinities can be significantly improved (often, at least an increase of 3-4 orders of magnitude is required).

By recognizing that there should be or may be changes (for example, where additional points should be added, the information of which may be provided by some embodiments of the present invention), it can be seen whether it is possible for one specific leader to undergo the required changes, for example, so that Have the intended attachment points in the correct positions. Specific meters (for example, which are connected) may indicate what changes are required.

These methods can also be used to reject some leader modifications provided during the leader improvement process.

It should be noted that some methods of deflecting a leader do not really require all possible meters and / or triangles to be suitable. Rather, even part of the library is suitable, for example, to reject some leaders. In one example, an incomplete coverage library is used that creates an incomplete map (e.g., part of the space, disconnected and / or not all binding points), which can be used to reject some leaders and / or indicate the potential suitability of others and to search. In addition, even the linking or failure of linking one meter may indicate the suitability or unsuitability of the leader. Usually, due to the uncertainty inherent in all chemical processes at present, decisions are not made based on a single binding study.

8.7 Target Mapping

In some embodiments, the binding of the meters is investigated during the search process. In one example, binding is used to test theories or assumptions about leaders. For example, if it is believed that some leader is suitable, then it can be assumed that at least one of several specific meters is associated. Leaders can be ranked, for example, based on how good such target binding is. Alternatively or additionally, part of the layout may be re-mapped as a result of the search process. For example, a search method may indicate conflicting facts about the layout structure. In another example, a higher resolution mapping of part of the layout may be required, for example, to more accurately determine the distance between two groups. In some cases, instead of conducting a study with a full range of meters, meters are selected based on their best ability to bind (or not) to specific parts of the layout. For example, if you want to determine the distance between two layout points, select meters that have less ability to communicate with other layout points. In another example, more specific groups are used, for example, having a more limited assortment of chemical behavior and / or have a greater focus. This may be required when using a different frame. Probably, the meters used for this re-mapping have fewer triangles per meter, for example, between one and three, to reduce the likelihood of unexpected binding. Alternatively or additionally, the meters are selected so that spatial inconsistencies prevent binding in undesirable places. In some cases, such meters are not in the base mapping library.

- 32 010258 definition used for the initial layout determination. In some cases, the required meters are synthesized specifically, and not selected from an existing library.

8.8 Checking target suitability

In an illustrative embodiment of the present invention, a map is used to determine the suitability of a target to be a target for a drug substance. The degree of suitability may be, for example, binary, or it may be graduated (discrete or continuous). In some embodiments of the present invention, the fitness value is not a scalar value, for example, a vector with each element of the vector indicating a different fitness aspect. A similar structure can be used to indicate the suitability of leaders and potential drug substances.

In one example, the use of target fitness testing occurs where there are numerous potential targets. For example, in some diseases, it is possible to choose among a variety of target proteins or to select different parts in the protein synthesis chain (for example, transcription of DNA, creation of a protein chain, folding, protein, subsequent processing of the protein and protein deployment). Some of these potential targets may not be suitable.

In an illustrative embodiment of the present invention, the map can be analyzed to find such suitability, for example, by deflecting targets with an active region that is too large (for some types of processing). The size of the target area can be found from the layout geometry. Alternatively or additionally, the target may be supposedly unsuitable due to the fact that it usually has a too active (non-specific) region of the target, which can be determined, for example, by analyzing the specificity of a particular target arrangement. Alternatively or additionally, the target may be presumably unsuitable due to its active region, which has a very weak affinity (for example, large molecules of the drug substance with many binding points may be required). Alternatively or additionally, the target may be allegedly unsuitable due to its similarity to an auxiliary protein. Such similarity can be determined by comparing the layout of the target with such known auxiliary proteins. Similarities to any human protein can help determine potential side effects well in advance. When classifying leaders, a leader can be assessed based on the possibility of its interference with an auxiliary protein, which is not necessarily determined by checking the binding of the leader to the configurations of auxiliary protein models.

In an exemplary embodiment of the present invention, an auxiliary protein layout database is provided. Such a database may be provided using methods known in the art. Alternatively or additionally, at least a portion of the database is provided by systematic mapping of auxiliary proteins.

Alternatively or additionally, at least a portion of the database is provided by creating the geometry of the target region of the most unfavorable variant or range of possible geometry for the active regions based on knowledge of the structure of the substrates that are activated by protein. Such target areas of the most unfavorable option can also be used as preliminary information to facilitate the decision which of several reconstructions is correct.

8.9 Target Sectioning

In an exemplary embodiment of the present invention, a map is used to identify portions of the target as potential exact targets, and on which the drug search method can be focused. Although the target, as a whole, is such that it is exposed to a drug substance, it can be affected in many ways, for example, different drugs can block different parts of the active region. Alternatively or additionally, certain drugs may cause conformational changes. Alternatively or additionally, some drug substances may interact with control regions on the target. Alternatively or additionally, some drugs may be agonistic, while some may be antagonistic. Alternatively or additionally, some binding regions may be suitable for use upon attachment (for example, as a base for the attachment of a molecule closer to the target region) rather than directly to the activity. The binding regions can be classified based on the type of exposure that can be assumed from the binding of the molecule to such regions. Such a classification can, for example, be done manually. Alternatively or additionally, an automatic classification may be provided, for example, based on a sample of the structure (for example, which indicates for a particular class of protein which region of the protein may be suitable).

Optionally, potential control regions that can alter the target are identified. Perhaps such control regions are identified based on binding in binding studies. Optionally, a target model is used to investigate whether binding can cause conformational changes in a potential controlled site, for example, based on the similarity of the sites of interaction on different adjacent parts of the protein.

- 33 010258

In an illustrative embodiment of the present invention, the active regions are segmented into different precise targets based on subregions that have a potential drug interaction, for example, based on their geometry. Alternatively or additionally, segmentation is based on the selection of such subregions that are not common to similar subregions of auxiliary proteins (e.g., segmentation in specific and general binding regions).

8.10 Analysis and improvement of the drug substance and leader

In an illustrative embodiment of the present invention, the above arrangement is used to analyze existing drugs and drug leaders, for example, to help improve or re-develop a drug substance or for screening.

In an illustrative embodiment of the present invention, the arrangement is used to determine which plurality of target regions on a target interact with a drug substance, or which target among a plurality of possible targets interacts with a drug substance. Such a methodology can be used, for example, to analyze the effect of drugs whose working method is unclear.

In another example, a drug substance is analyzed to determine how much of the drug substance binds to a target. This can serve as the basis for a drug modification process in which parts of the binding of the drug substance are retained, and other parts of the drug substance are modified. Alternatively or additionally, when modifying a drug substance, care must be taken not to deform the active part of the drug substance so that it does not bind, or to deform the drug substance completely so as to cause spatial inconsistencies.

It should be noted that one drug substance can interact with two different targets in the desired way, with each target interacting with different, possibly overlapping parts of the drug substance. Such activity of a drug substance is not necessarily determined by comparing the structure of the drug substance with the structure of the targets.

In some cases, the exact spatial and chemical properties of the drug substance (or protein substrate) are not known. However, by determining the layout of targets that bind to the drug, the spatial and chemical composition of the active part of the drug can be estimated.

In another example, the arrangement is used to determine the pharmaceutical activity of synthesis by-products. Upon receipt of a drug substance using a specific method, various by-products are also formed, some with beneficial activity, and some with unfavorable activity. In an illustrative embodiment of the present invention, the structure of such by-products is compared with target regions and auxiliary proteins, taking into account the assessment of what side effects they can lead to. The method for producing the drug substance is not necessarily selected or rejected based on the activity of the by-products evaluated in this way, and it has been found that the type and quantity of by-products produced by a particular method can be determined. Alternatively or additionally, such a comparison can be used to facilitate the improvement of the production method and / or when deciding which synthesis parameters to use. Such testing can also be used for regulatory purposes, for example, to approve or disapprove of generic drugs.

8.11 Drug selection

In many cases, there may be many drugs that can cure diseases. Knowing which target (and auxiliary proteins and / or other human proteins) is exposed to drugs, and how it interacts, can be useful when choosing between alternative treatments, to prevent side effects, prevent drug interactions or control them and / or when choosing the treatment of diseases for which it is impossible to select the exact medicinal substance, for example, exotic tropical diseases and some viral diseases.

In an illustrative embodiment of the present invention, the target arrangement is used to select which of the many available drugs or drug leaders appear to be most suitable for interacting with the target. In the case of drugs, this may allow the selection of alternative treatment protocols. Also, in some cases, knowledge of the interaction method may facilitate the selection of such moments and / or related protocols and / or combinations of drugs in which the drug is most effective and / or has minimal side effects.

Alternatively or additionally, drug substances can be designed to interact with a large number of targets. For example, a leader that interacts with a large number of targets (for example, some other disease or syndrome) or parts of the target area may be rated better for further processing than other leaders.

- 34 010258

A possible relative application is to find a new application for an old drug substance and / or to facilitate the determination of how to modify an old drug substance for a new application. For example, when searching for leaders that match a sample, you can also search through a database of drug substances to see which drug substance has a structure that is predicted by the modeling process to provide good binding. In general, existing medicinal substances have other properties (AOMET).

8.12 Drug Improvement

As noted above, knowledge of the mode of interaction and / or the problem of interacting with the target region can help in modifying the leader to become a drug. Alternatively or additionally, such knowledge can be applied for use in improving an existing drug substance and / or modifying a drug substance for interacting with a target relative to an existing target. By comparing the layouts of the two targets, for example, possible suitable changes in the drug substance can be determined. Alternatively or additionally, the layout of the target can be used to study the problems of binding of the drug substance to the target (for example, very strong or very weak) and / or to determine the effect of drug modification on this binding behavior. In an illustrative embodiment of the present invention, the potential drug substance, if bound, is examined in relation to a model to determine if there are groups that can theoretically be added that can contact another point in the binding region.

Alternatively or additionally, an improvement in a drug substance comprises an improvement in a drug substance to match more than one target or multiple target mutations, for example, comprising one group for binding for one mutation and one group for binding for another mutation, for example, in HIV, some proteins have two main species and numerous subspecies. Such an enhancement may interfere with other properties of the drug substance, but a compromise solution may be considered useful.

Alternatively or additionally, a drug substance can be designed to bind to a subset of binding points that are common to multiple targets or mutations, for example, analyzing multiple target models to determine the shape of the binding points. Various drug search methods are then optionally used, assuming that only these binding points exist. A real study of a potential drug substance can be performed on a variety of targets to ensure that various modifications of the drug substance will not make it unsuccessful to bind to one of the targets. Alternatively or additionally, if a modification is made, it is determined whether the modified drug substance can bind to common binding points and / or whether there are spatial inconsistencies. It should be noted that there may be other reasons for searching for a drug substance that only binds to a subset of possible binding points, for example, if a mutation is expected at one of the binding points and / or to allow the drug substance to work, even if an interfering molecule binds to one of the points binding.

8.13 Failure to analyze and re-develop a drug substance

Often a drug substance can be put on the market and then fail. The method described herein may be suitable for determining the cause of failure, and then possible assistance in the rehabilitation of a drug substance. In an illustrative embodiment of the present invention, a target composition of a drug substance and / or other proteins with which the drug substance is supposedly interacts is created (for example, based on the type of side effects). Then, the drug substance is compared with targets to determine failure in binding to the correct target and / or undesired binding to non-targets. It should be borne in mind that although such a comparison can be theoretically possible using other means, it is assumed that before the possibility of mapping the target, such a large scale of displaying the active regions of the targets was not advisable due to the time and cost limitations.

In an alternative embodiment of the present invention, it is noted that the drug substance may be suitable only for part of the population, for example, due to individual differences. In an exemplary embodiment of the present invention, genes that express false targets and / or targets are used to reconstruct target models or samples, and then display the active regions of the models. The results may show that the individual is sensitive to the drug substance and / or that different individuals are resistant to the effects of the drug substance. Alternatively or additionally, testing can be performed on pathogenic lines to determine different susceptibility to drug substances. In some cases, genetic differences are associated with already known markers, for example, sensitivity to sulfates is associated with a C6RE deficiency for sulfates, so that the classification of people based on drug compatibility can be simple. Alternatively, prior to choosing which medicinal substance to use, genetic

- 35 010258 test.

8.14 Additional analysis related to drug discovery

Additional methods of analysis can also improve the method of searching for drug substances. For example, many medicinal substances have side effects due to their interaction with auxiliary proteins or proteins that cause a feeling of dysphoria if interference occurs with them. Examples include 01 proteins and liver proteins. Some drug targets are known as similar to such proteins. In an illustrative embodiment of the present invention, models are created for such potential generators of side effects. Any potential drug leader is rejected (or rated lower) if binding to one of these prohibited models is indicated. Alternatively or additionally, drug substances that have known side effects are analyzed to determine which protein they bind to, and such protein and / or specific binding sites are used to determine the inhibition of binding of the potential drug substance.

In another analysis example, potential drug molecules are analyzed to see if they bind as a substrate to certain enzymes. Such binding may indicate the rate of failure of the drug substance or its excretion. Alternatively or additionally, such binding may be useful for identifying prodrugs that activate by interacting with certain enzymes, such as liver enzymes. In such a case, the drug substance may include two sets of target regions, one for activating the drug substance and one for binding the drug substance to its target. Optionally, binding to a protease (or other manipulation protein) is guaranteed by adding binding groups or gauges to the drug molecule in suitable places.

In another example, a set of target molecules that is known to be exposed to the same protein or molecule is analyzed to determine if they have a common binding geometry to which the molecules bind. This can help, for example, when finely tuned, the molecules bind more selectively, for example, by adding a group that will interfere with other molecules of the target and / or help with binding to a specific target molecule.

8.15 Directional drug search method

As can be taken into account, the search method usually involves going through various dead ends. In an illustrative embodiment of the present invention, target mapping is used to select parts of the search method that are suitable for rejecting or preventing them from being used in experiments. Some examples (some of which are described elsewhere in this description) include deflecting targets that appear unsuitable for improvement, identifying targets that cause side effects, and cleaning the library. In an exemplary embodiment of the present invention, purification of existing libraries is accomplished by removing leaders from the library that have an expected low probability of binding and / or exhibit excess to other molecules. For example, a molecule that is very plastic is less suitable for binding. The probability of binding can be estimated, for example, using reasoning in terms of energy, based on molecular degrees of freedom.

8.16 Creating a convenient model

Despite the fact that many proteins and molecules are organized into a catalog, many of them are not known convenient models. To determine the exact applicability of a protein or molecule can be very expensive. In an illustrative embodiment of the present invention, potential convenient models of molecules and proteins can be created on a large scale in the following way. The molecule may be a convenient model as a meter or it may be a convenient model as a leader or drug. In an exemplary embodiment of the present invention, existing locations of the target region, for example, 10, 50, 100, 1000, or any smaller, larger, or intermediate amount are matched to the molecule to see if binding is appropriate. It is believed that many molecules may prove to be potential convenient models. In general, more compliance is more workable and increases the likelihood of success.

In a similar method, protein mapping provides an indication of their forms of active regions, potential substrates and / or potential drug substances that can affect them. In an illustrative embodiment of the present invention, a convenient model is found for a protein by determining its substrate. Optionally, the arrangement of the active region of a protein is compared with the structure of known substrates and proteins.

In such a method, a library and individual drugs and proteins can be indicated as an expected convenient model. For example, a protein can be one of the following families of ORSK proteins, proteases, kinases, ion channel mediator proteins, or any type of protein or other macromolecule found in living organisms.

- 36 010258

9. Illustrative Search Applications

9.1 Overview

In this section, existing search methods will be described, as well as possible modifications that are taken into account in the methods described herein.

Despite the fact that many approaches to the search for drug substances are known, usually in the existing methods use two main following approaches.

9.2 Screening drug development

The drug search method works by screening a target for a relatively large number of molecules and then attempting to improve any matches for drug production. The process is as follows:

(a) A conventional library of screening compounds is provided that is equally relevant to all target proteins. The usual sizes of such libraries are constantly growing approximately by one order of magnitude (coefficient 10) per decade. The usual sizes are currently 110 million. Libraries are often privately owned and maintained by each corporation independently.

(b) A corporate library is screened for the selected target. A search is made for compounds exhibiting at least weak activity (significant activity at concentrations of typically 1-100 μM) of the type required for the target.

(c) If no successful hit is found, the process ends. Obviously, this happens often, in about 70% of cases. If a successful hit is found, the optimization stage begins, in which, as the final result, a compound with strong activity (at a concentration of usually nM) with respect to the target is expected. This is carried out by one or a combination of the following two methods:

1. If there is only one hit, or if all hits are variants of the same molecular base, a large number of analogues of such a hit are synthesized. Such a group of compounds is sometimes known as a focused library. They are also screened for the target protein. The goal is to determine the direction for increasing the activity of the initial hit by identifying the chemical groups and positions on the initial hit, the activity of which is increased. Such a method is known as developing (quantitative relationship of the activity of structures).

2. If the number of chemical groups is identified as hits, the computational process for identifying possible pharmacophores (molecular structures directly involved in binding to the target) is performed. They can indicate not only possible optimization directions, but also their feasibility for a given molecular starting point (both from a physical point of view and from a synthesis point of view).

(b) A by-product of such a process is usually a drug substance of suitable quality. Molecules in the source screening library are often selected to obtain drug-like properties. During the optimization process, only part of the information is available in such a way that at the same time requirements for compliance with drug similarity and increased activity are rarely directly controlled. The final drug candidates that may result from this process have exact resemblance to the hit compounds in the original screening library.

(e) Testing. Candidate drug substances are tested, for example, on live animal models and then on humans to determine efficacy. Many drug candidates are unsuccessful from this point of view and, in the absence of any basis for modification, are completely inadequate.

In an illustrative embodiment of the present invention, the above invented methods can be used to improve the above method of searching for drug substances, for example, one or more:

(a) Hit ratio. As stated above, in most cases no hits are found for a new target. By creating target mapping, leaders used for screening can be selected better. Even leaders with very low affinities can be selected for further improvement thanks to a combined indication of very low activity and matching cards. Alternatively or additionally, a method for developing a meter library is applied to the molecular library to reduce repetition and to facilitate guaranteeing coverage of the binding space. This can be done, for example, by analyzing the library to identify meters in the space of triangles and / or the uneven distribution of leaders in this space. Additionally, excessive overlap can be determined. Alternatively or additionally, the library can be analyzed to determine molecules that are not even suitable for binding, for example, because of their excessive flexibility and unknown method of binding. Alternatively or additionally, if screening exists in steps, then molecules for each step can be selected based on their less overlapping.

- 37 010258

Alternatively or additionally, some binding results may not be taken into account, for example, a molecule with high flexibility can add a lot of noise (binding to many molecules in many ways) and, therefore, not taken into account at least in the first stage of processing.

Alternatively or additionally, meters that bind can themselves be used as leaders (and a large number of such bindings are contemplated). Often the meter library is small compared to the corporate library and can be added to it with relatively little inconvenience. In an illustrative embodiment of the present invention, the results of the old library can serve as starting points for optimization (as before), but the optimization can be directed using information obtained from screening using meters. Probably, the study of the binding library of meters is performed on a target with an interacting leader. This type of study can be used to determine whether the leader (or molecules from the library) interacts with the active region or not (for example, on the basis of whether this affects the binding of the library of meters, and the magnitude of this effect). Such a study may be comparable to a study performed with other binding leaders and / or with completely unrelated leaders. The influence of the chemical structure of the leader can be determined by checking the study for the presence of one or more chemically similar, but not interacting leaders.

(b) The direction of the process. If target mapping is done and the starting point is known, there are still many ways to improve the leader in drug production. In an illustrative embodiment of the present invention, knowledge of the target geometry and / or chemical behavior is used to assist in the direction of the modification process, physical substitution experiments with virtual targets, and / or help in selecting (possible) useless leaders. Additionally, it was noted that different combinatorial creation of leader modifications can be simplified by selecting only those leader modifications that are significant (or are most significant) in terms of target layout and / or based on the three-dimensional structure of the leaders (for example, by checking which triangles manifest themselves with the help of which leader and with the help of which modification of the leader). Optionally, a mismatch between results based on a particular layout and the actual activity of leader binding may be useful in correcting the layout, a better understanding of the leader’s chemical structure, and / or prediction of other leaders that may be promising.

(c) Recovery of a drug substance. Even if the drug substance has not passed the testing step, in an illustrative embodiment of the present invention, the above methods can be used to determine the causes of failure and / or provide guidance on the modernization of the drug substance.

9.3 Screening alternative drug development

Chemical genomes or chemogenomes have recently become very popular. They are based on the idea that instead of the first search for a target and the subsequent search for a compound, the opposite method is used for it: first, compounds for screening are examined for the whole cell, looking for a phenotypic result (for example, the selective death of cancer cells). Then, after finding the active compound, a target is sought. One of the likely advantages of this approach is the parallel operation on a large number of targets, many of which may even be unknown. However, existing screening libraries cannot guarantee hits. In an illustrative embodiment of the present invention, a library of meters is used as described herein, and it is contemplated that there are many meters that interact with cells. Since interactions can be weak, a non-trivial number of such interactions can be expected.

9.4 Development of a drug substance based on the structure

This method assumes that accurate simulation software is used to reproduce the molecular process. The method is as follows:

(a) Obtaining an accurate and detailed three-dimensional structure of the target protein. Usually carried out by x-ray crystallography or NMR analysis (both experimentally). There is also a computational approach, but it is usually not accurate.

(b) Identify active sites in the protein structure (not always in the direction of new, unknown targets).

(c) Identify suitable binding points in the active site, as well as points known as pharmacophores. These are the points at which weak (non-covalent) binding occurs.

Potential ligands must correspond to the number (usually 6 or more) of such points at the same time to achieve nm tools.

(i) Molecules are developed that correspond to the active site, both geometrically and in terms corresponding to a sufficient number of pharmacophore points. Both this step and the previous one are performed using linkage or molecular molecular modeling

- 38 010258 mechanical type.

In an illustrative embodiment of the present invention, the proposed methods described in the present description can be used to improve the above method of searching for drugs, for example, one or more of the following:

(a) Related structures.

3Ό protein structures are obviously in many cases of little benefit in themselves. Many studies have shown that there are difficulties in developing strong ties, if only based on such (i.e., geometric) information. In an illustrative embodiment of the present invention, it is noted that useful information is in 3Ό target structures associated with ligands. Since such ligands are originally known, in an illustrative embodiment of the present invention, meters that bind to the target are used instead of such ligands, with the assumption that a significant number of such meters for binding can be found. In an illustrative embodiment of the present invention, the binding process of the meters is used and then the target is simulated (for example, using NMR or X-ray crystallography), possibly several times, with different meters connected. It is believed that the shape of the target region with attached meters is suitable for developing strong bonds using methods known in the art. Possibly, the known methods can be modified, for example, to collect the results of different configurations caused by different binding sites of different targets. Optionally, providing a plurality of binding meters (e.g., 5, 10, 25, 50, 100, or any smaller, intermediate, or larger number) may help determine the target binding method (s), possibly improving understanding also by providing private binding methods. In general, providing more meters means more work, but can improve the accuracy of the analysis.

In an illustrative embodiment of the present invention, related structures obtained from a plurality of meters are combined, for example, by overlaying with a target as a base. Such an overlay can result in a general model of the binding region of the target and / or a fully connected configuration, rather than parts of it that can be provided by each meter.

(b) Comparison. In an illustrative embodiment of the present invention, the shape of the active region defined by the simulated model is compared with the shape of the region as determined by the mapping method. The differences between the two methods can help with the correction of the mapping / reconstruction method or with its correction in modeling the model. An optionally simulated model is used to choose between alternative reconstruction and / or to facilitate fine-tuning the reconstruction, for example, by facilitating the calculation of more accurate distances and / or by indicating which possible groups may be involved in binding.

(c) Identification of binding points. In general, software modeling is not accurate enough to predict the binding points in the target protein. Active areas can also be difficult to identify. This is especially the case with new targets. In an illustrative embodiment of the present invention, the above methods circumvent one or both of these problems by experimentally identifying potential binding points and / or methods, for example, using a standard library for examining meters. Then the active regions are analyzed with greater depth, using software, for example, to predict the affinity of new compounds with a specific target.

9.5 Modular assembly of ligands

This method, which is obviously used by Ziyeik ts., Works by constructing leaders from parts that show affinity. The method is as follows:

(a) A final library of elementary molecular fragments is synthesized that includes a linker port (i.e., a portion of a molecule in which attachment can be easily performed). These are usually small molecules, previously identified as pharmacologically interesting, and which are susceptible to the inclusion of a standard linker port.

(b) Screening of elementary fragments for the target protein, looking for extremely (~ 1 mm) low affinity. This step is usually problematic.

(c) Link groups of two or more fragments through their linker port components to achieve increased affinity. The distance between two fragments, i.e. binding chain length, can vary and optimize.

In an illustrative embodiment of the present invention, the disclosed methods set forth in the present description can be improved by the above method of searching for drugs, for example, one or more of the following:

(a) Elementary fragments are not currently developed in the art using any logic that can be considered exhaustive, i.e. conventional scatter metrics are used (as in standard screening libraries), but they are not compiled into the final

- 39 010258 list. Therefore, hits are rarely found (for ordinary targets), even less than for conventional screening libraries, possibly due to the very low expected affinity, which formulates many technical problems (for example, solubility). In an illustrative embodiment of the present invention, a set of fragments is selected based on the coverage of the space. For example, fragments may be pairs (triangles) of groups having distances, and group types are selected to cover the probability space.

(B) Geometry, i.e. the characteristic distance and orientation between two weakly binding groups is completely absent from the results of the original screening library in the art. At the linking stage, only very limited geometry variants (i.e. linker length) can be tested. In an illustrative embodiment of the present invention, linking a library of gauges is used to provide geometric hits (or a complete model) that helps in deciding how to connect fragments, which fragments to connect, and what distances to set between fragments. It can also help in determining which type of linker to use when connecting fragments. It can also be used to synthesize a new molecule that includes the binding parts of the binding meters oppositely spaced by a suitable structure (for example, a change in a known drug substance).

10. Illustrative non-search applications

The above measurement methods can also be used for other applications than the search for drugs. For such applications, a different set of meters may be required.

In an illustrative embodiment of the present invention, measurement methods are used to evaluate toxicity, for example, to identify auxiliary proteins that may have adverse interactions with some drug substance or potential toxin. This may be useful in determining the toxicity of industrial or household chemicals.

In another illustrative embodiment of the present invention, measurement methods are used to predict the affinity of an antibody for a substance and / or cell, for example, by identifying binding sites on an antibody and / or substance.

In an illustrative embodiment of the present invention, measurement methods are used to display the external side of an organism, for example, a virus, rickettsia bodies, a worm, a single-celled protozoa, fungi, amoeba or bacteria. It may also be useful when developing a vaccine. For example, a vaccine is often more effective if it is derived from a protein whose form is not changed. By determining which parts of the binding regions on the outside of the pathogen are not changed, such a determination can help in selecting a particular protein from the pathogen for use in vaccination and / or in assessing changes in the development of a suitable vaccine. To prevent autoimmune responses, the active regions of an existing vaccine substance can be mapped to see if the sample resembles sufficiently strongly that of real proteins. It should be noted that this correspondence may depend on the genetic material of the individual.

Alternatively, for absolute measurements in some embodiments of the present invention, the above methods are used to determine relative measurements, for example, to measure conformational changes in a protein under different conditions. Identical (or different — for example, to match new proposed measurements) binding studies can be applied to a protein under different conditions. Perhaps more flexible meters and / or less stable meters are used for such an application.

In another illustrative embodiment of the present invention, the aforementioned method is used to search for new agricultural chemicals, such as insecticides and herbicides, that are specific targets by exposure to proteins known as key only for certain types of parasites or weeds. Alternatively or additionally, artificial hormones are being developed to match targets in plant cells.

11. Use of preliminary information

In some examples, the above method is described as blind, which involves a neutral starting point with essentially no knowledge of the target. In some cases, there is prior knowledge of the target, collected from various sources and / or using previous measurements of the target. Such preliminary information can be used in many ways. Some examples follow.

In an illustrative embodiment of the present invention, background information is sufficient to suggest several alternatives. A study of linking with a library of meters with or without reconstruction can provide sufficient information for choosing between alternatives, for example, between alternative models, some of which have a leader interacting with the target, or choosing between two types of reconstruction of the layout of the target area. Optionally, for this purpose, the set of meters can be reduced to only those meters that may differ and / or are necessary for any one of the models.

- 40 010258

In another example, X-ray crystallography, NMR, IR spectroscopy and / or chemical properties of the target are used in the above reconstruction method, for example, to solve uncertainties and / or to compensate for the lack of data. In one example, such methods demonstrate how one or more meters actually communicate with a target. In another example, such methods or other prior knowledge is used to force some structure intended for reconstruction, rather than the subsequent reconstruction-based assessment indicated above. For example, forcing the structure to include some subform (for example, the tetrahedral part), which for some reason could not be reconstructed from the research data.

In another example, if a part of the target is known, it can react with a substrate that blocks such a known part so that the measurement can only be applied to the unknown part. Alternatively, interaction statistics in a known part can be used to help in binding statistics associated with a structure in an unknown part. For example, a computer model or similar target can be used to provide an estimate of which targets bind and with what force in a known part. When analyzing the results of a study, meters that bind to a known area are not taken into account, are not used in the study, and / or their binding strength is reduced during analysis. Optionally, the meter is not removed for reasons that removing it may not leave triangles of a certain size and / or group for binding in an unknown region. Alternatively, the library is fully utilized, for example, as noted above, that simultaneous screening using 100,000 studies at a time is a modern technology.

In another example, if an alternative measurement method is used, preliminary information may provide an understanding of the desired starting points.

Optional preliminary information is used as input to modify the binding method, for example, by changing the environment.

In another example, preliminary information is used to determine the environmental conditions used during the measurement, for example, using information from attempts at preliminary studies with a similar protein to indicate that environmental conditions are suitable for binding and / or at least do not interfere .

In an illustrative embodiment of the present invention, preliminary information is used to develop specific scaffolds, groups, and / or meters for conducting improved measurement of a specific target. Molecules can, for example, be specifically designed, and / or a sub-library constructed by selecting previously known molecules. In an illustrative embodiment of the present invention, a framework is selected for such a sub-library due to a small (e.g., 0.5A) difference in the side of the triangle due to a change in the framework. In a conventional mapping method, this difference may not be important, but in high resolution mapping, for some targets (for example, where there is poor binding) this may be important. Similarly, a set of meters can be provided to cover a certain range of sizes and / or types of high-resolution chemical behavior.

12. Iterative measurement

In some methods that are adequate for using preliminary information, iterative measurements make it possible to generate information from the preliminary measurement stage for use, for example, to better tune the current stage or to reject some probabilities.

In some embodiments, the implementation of the present invention, in addition to the method of measuring one step, for example, as described in some embodiments, implementation above, use the method of iterative measurement. In one example of such a method, a lower resolution reconstruction is created. Then conduct additional research using the same or different library of meters, and provide reconstruction with a higher resolution. An earlier reconstruction may be used, for example, as a starting point for a reconstruction method and / or to facilitate the selection of which meters to use in an additional study. In an illustrative embodiment of the present invention, such an iterative method is used if the cost and / or time to complete one study is completely greater.

In an illustrative embodiment of the present invention, the iterative measurement uses more flexible meters (explained below) in the first measurement set than in the second measurement set. Alternatively or additionally, different subsets of meters are used for different sets of measurements.

The differences between the stages can be in the correctness of the reconstruction, for example, which ones, where the places of interaction lie. Alternatively or additionally, the difference may be in accuracy, for example, in the distance between two binding sites or the angle of communication of the binding site. In an illustrative embodiment of the present invention, the above range coverage assumptions, for example for hydrophobic bond sizes and for directional bonds, are made stricter in later reconstruction iterations, for example, providing 15 directional bonds. However, not all measurements

- 41 010258 may be necessary for repeated execution. Instead, only those meters are used that are associated with the places of interaction that are supposed to be changed in the model. Various search methods are used, known in the art, to facilitate the provision and / or determination of the convergence of the study and reconstruction method, for example, searching for an extremum.

13. Meters, physical properties

13.1 Overview

The various applications of meters are described above, some of them may use a complete library of meters (for example, fully encompassing and having sufficient resolution) and some of them may alternatively or additionally use an incomplete library. One or more of some studies are not necessarily considered when developing such libraries. Such illustrative studies and solutions that may not necessarily be applicable in the design and / or selection of meters, meter designs, and / or meter sets are described below. As noted, some studies relate to the properties of individual meters, and some to the properties of meters as a set. The development (and / or selection) of a complete set of meters can be addressed to numerous studies and various alternatives, for example, as shown in the illustrative set of meters described below. Such studies are explained below. In the general case, it should be noted that even some of the meters in the set of meters are not suitable, this usually does not detract from the suitability of the entire set of meters.

In FIG. 4 illustrates an exemplary meter 400. A typical meter set includes a large number of meters. Perhaps all meters are part of the basic overall development, and can also be described below, but this is not essential. Additionally, there may be a large number of meters, meter designs, and sets of meters that are suitable for measurement.

In an illustrative embodiment of the present invention, a significant portion of the set of meters is based on changes in a small number of basic molecules called frameworks. In this development method, the framework includes a set of attachment points, and each meter is designed by selecting a framework and placing different groups at the attachment points. One of the potential advantages of such an attachment is that some different chemical methods are required to synthesize the library. Another potential advantage is that the created library has a more predictable chemical behavior, reflected, for example, in the environments used for research. Another potential advantage is that a more predictable and / or variable set of distances between groups can be achieved. Another potential benefit is the simplicity of developing an embracing library. Another potential benefit is that it is easier to guarantee coverage in the library or part of the library. Another potential advantage is that the use of these types of changes (possibly with wireframes new to the library) supports the creation of missing or desired dimensions, specifically. In one case, for example, new meters with specific distances are created by modifying the existing frame. It should be noted that not all such potential benefits are contemplated in each embodiment of the present invention.

It should be borne in mind that for a given library, parts can be based on frameworks, since other parts are created using other substances, for example, selecting from an existing molecular library and / or constructing using various molecular constructs using development and synthesis methods known in this technical field, trying to estimate the cost of developing molecules with specific properties. In addition, an entity library may not be based on wireframes. It should also be borne in mind that all framework-based libraries provide all, some, or even any of the above benefits.

13.2 Wireframe.

In FIG. 4, meter 400 includes a chassis 402 to which four groups are attached to four of as many attachment points as possible. In an illustrative embodiment of the present invention, meters 400 are selected to cover a range of distance between groups. In an illustrative embodiment of the present invention, by changing the location of the group link among the available attachment points, various intra-group distances are set for one meter. A larger range of possible values is not necessarily achieved by providing a range of possible frames. However, it should be noted that the frame itself is not required. Rather, it is expected that, at least for some embodiments of the present invention, it may be more cost-effective to create a library combinatorially using frameworks. This is illustrated in FIG. 4B, where the meter is shown as a triangle defined by its groups and the distance between them without any reference to the creation of the frame.

However, in an illustrative embodiment of the present invention, a framework is provided from which many different meters are constructed. Many different or identical groups can be selectively attached to different places on the frame using relational

- 42 010258 relatively standardized methods for combinatorial creation of chemical compounds, such as creating a range of meters that may have well-known chemical properties (e.g. solubility, saturated vapor pressure, stability).

In some embodiments of the present invention, the frame (s) is selected so that it does not extend to the borders or go beyond the boundaries of the size (sizes) of the triangle using groups. Alternatively, or in some cases, the framework and / or some groups actually interfere with the binding and may cause spatial mismatches. By providing a range of frameworks, spatial inconsistencies of some meters can be avoided and / or the causes of spatial inconsistencies can be determined.

In some embodiments, implementation of the present invention, the geometry of the frame and / or chemical composition are significant.

Optionally, the participation of the meter in ensuring the binding of the triangles is not taken into account when developing a set of meters. Alternatively, the reactivity of the framework is taken into account when developing the kit, for example, to provide one or more groups. Optionally, the influence of the framework on the provision of binding, repulsive and / or interfering bonds is taken into account during reconstruction or analysis. Alternatively or additionally, the geometry of the frame is selected for consideration in the analysis, for example, to determine the causes of spatial discrepancies.

Alternatively or additionally, the analysis of the binding triangles ignores any binding of the triangles that may not be found for the triangle (for example, based on the geometry of the meter).

13.3 Volumetric geometry of meters

As a rule, triangles define a plane, which may or may not be the plane of the frame (let it be any). In an illustrative embodiment of the present invention, if meters are selected for inclusion in a library, they are selected so that the attached groups lie in a plane or in some other desired correspondence. Plane placement has the potential advantage of preventing the incorporation of multi-stable (eg, conformationally variable) molecules, which is undesirable for some embodiments of the present invention, as they can interfere with analysis and / or reduce the likelihood of binding. Perhaps a set of meters is provided to cover the range of possible non-planar orientations. In some embodiments, this is more desirable than selecting molecules that exhibit conformational changes. Molecules with conformational changes can be excluded using other methods, as well as, for example, by analyzing each potential meter. Alternatively or additionally, the gauges are selected so that the dimensions of the gauges or specific triangles in them do not change, even if other parts of the gauges show conformational changes. Optionally, a certain triangle in the meter can be neutralized by making it energetically unsuitable for binding, for example, by ensuring that the triangle shows conformational changes or adding flexibility to the bonds of one or more of its groups. However, it should be noted that such a precise modification of the meter may not be possible, for example, due to the small size of the meter or its possible effect on other parts of the meters and / or other triangles.

13.4 Flexibility

The flexibility of the meter can adversely affect one or both values of the information provided by the correspondence of the meters and the affinity of the meter to the target. Since it is reliable that flexible molecules are more suitable for finding suitable binding points, increased flexibility may, at least in some cases, generally reduce the possibility of binding of the molecule for entropic reasons. In addition, the binding of a flexible molecule provides less accurate information than the binding of a rigid molecule.

Thus, although using a flexible meter, a larger number of layouts of the interaction points may be appropriate, in an illustrative embodiment of the present invention, at least some relatively rigid meters are selected for the meter library so that measurements using such meters are more accurate. Optionally, substantially all of the gauges in the gauge set are substantially rigid.

In an illustrative embodiment of the present invention, the meters are rigid in terms of translation, since the distance between the groups does not vary significantly. Alternatively or additionally, the meters are rigid in terms of rotation, since the relative orientation of the groups does not change. Optionally, the flexibility extends to the chemical specificity of the groups, for example by selecting groups that are more or less specific. For example, you can choose groups that have only one function (i.e., for the selected hydrophobic tert-butyl or non-aromatic ring (e.g. cyclohexane) or for hydrogen bonds, avoid using hydroxyl (OH), which is both a donor and an acceptor) or vice versa.

However, in the illustrative embodiment of the present invention provides a small degree

- 43 010258 flexibility, for example, to guarantee overlap between meters. In one example, the degree of flexibility is sufficient so that a pair of groups in the target can be matched using multiple pairs of groups in the meter with different distances between them. In an illustrative embodiment of the present invention, the meters are designed so that each distance between the groups in the target can correspond to both a meter that has a slightly larger distance and a meter that has a slightly shorter distance. The degree of flexibility can be determined so that a relatively low amount of energy is required to bind or stretch the meter so that it can match the arrangement of groups in the target. Relevant energy levels may depend, for example, on the sensitivity of the studies, on the concentration of the meters and / or the research environment.

Optionally, at least a small number of meters is flexible, for example, to compensate for meters that are unsuitable. For example, as noted herein, flexibility with respect to rotation may allow the participation of hydrogen bonds and / or aromatic rings. Alternatively or additionally, flexible meters are used to facilitate providing low level information, which can then be finely adjusted using rigid meters. An optionally reduced amount of information (for example, by lack of linking and / or less accuracy) is compensated by the excess of meters and the units of triangles in the meters.

It should be noted that the specific way to determine which triangle is connected, described above, provides significantly more weight for rigid triangles. It should be noted that in one meter triangles can have different degrees of stiffness.

In an illustrative embodiment of the present invention, the software Ca1a1uz1 from Lcse1gu§ (formerly M81) is used to analyze the meter stiffness.

In an illustrative embodiment of the present invention, at least 20, 40, 60, 80%, or any smaller, intermediate, or greater percentage of meters are rigid. In general, if stiffer meters are used, they facilitate analysis using the methods described herein. However, such meters may not be suitable and / or for various reasons may be desirable for the use of non-rigid molecules, for example, if such molecules are suitable medicinal substances or have other properties useful to make them suitable for screening.

In an illustrative embodiment of the present invention, a substantially rigid molecule (or bond) is defined as a molecule that has one entropy configuration and in which, except for hydrogen atoms, there is no bond movement of more than 1A when using less than 20 kcal / mol. In alternative embodiments, implementation of the present invention can allow less rigidity, for example, allow more movement, such as 0.8A, 1.5A, 2A or any larger, smaller or intermediate value at 10, 15, 30, 40 kcal / mol or any smaller , an intermediate or greater application of energy. It should be noted that absolutely rigid molecules are usually impossible. Instead, the term substantially rigid is used in the claims. As molecules become less rigid, they can bind with greater difficulty and less specific in the sense of their binding. However, less rigid molecules can be more easily obtained and / or used for guaranteed coverage, for example.

Typically, rigid molecules are those in which all single bonds are either part of a ring or attach terminal atoms, i.e. to one of their ends (for example, single atoms or simple groups, such as ΝΗ ₂ , for which rotation is not of interest in some cases). As soon as the ring grows too strong, for example, more than 5 or 6 atoms in some cases, the ring becomes flexible. Rings with large sizes can also be rigid, for example, if there are no more than 2 adjacent single bonds, the atoms of which participate together only in single bonds (i.e., if any of the atoms in the ring themselves are joined by a double bond to an atom that is not a member of the ring, it can also provide rigidity for such a segment of the ring). A single covalent bond is free in terms of rotation, unless it is part of a ring.

13.5 Meter lengths

In an illustrative embodiment of the present invention, the lengths of the sides of the meters (i.e., the distance between the center of mass of the groups) are chosen so as to cover the range of expected distances between the places of interaction and / or the size of a small molecule of drugs. Alternatively, for example, for large molecules of drug substances, a different range can be chosen than for small molecules of drug substances. In an illustrative embodiment of the present invention, the selected range is from 2 to 12A. In another example, the range does not exceed 10A or does not exceed 8A. Alternatively or additionally, the range is greater than about 3A or 4A. In some cases, the outer length or inner length may be useful, which is determined by the participation of the outer or inner groups in the triangle.

In an exemplary embodiment of the present invention, a sample is selected such

- 44 010258 at a time to get a uniform sample of the energy costs required by the molecule to match the sample resolution. For example, if the first side of the triangle has a length of xA and the second side of the triangle has a length of A, the range of distances covered by the first side should require the same amount of energy to modify the molecule to fit the range as the range of distances covered by the second side. This usually means that since the molecule is large, the binding range for the same amount of energy increases. An optionally allowed energy value is a parameter of the method for investigating the target and / or meters used, for example, to allow the binding found by the meters.

In an exemplary embodiment of the present invention, the range is covered in intermediate sizes so that at least one meter can correspond to each intragroup distance for each pair of groups. Alternatively or additionally, at least two meters or sides of the meter are similar in group geometry. Alternatively, only two sides of the meter correspond. A different external environment may impose a different number of meters, for example, some bonds may show greater flexibility at one temperature, but not at another.

The selection of distances with the help of meters can even be in accordance with the range or can vary, for example, exponentially and / or stepwise, due to the influence of changing frames between the triangles to achieve different lengths of the sides of the triangles.

It should be noted that some sets of side lengths cannot be combined into one triangle due to the required relationships in the triangle, namely, the sum of the lengths of any two sides is greater than the length of the third side.

13.6 Environmental Stability

In an illustrative embodiment of the present invention, the meters are used for the target under normal physiological conditions, including controlled pH, temperature, ion content. Thus, they can only be selected correctly executed in standard environmental conditions.

However, in some embodiments, the test range may not be consistent with generally existing physiological conditions. In a specific example, the desired property of the drug substance may be activity at hyperthermia temperatures or in patients with fever and abnormal physiological temperatures.

A specific set of meters can be used for non-physiological conditions, for example, replacing some meters with others. Alternatively or additionally, a relatively stable set of meters can be provided that exhibits the same behavior over a wide range of environmental conditions. Alternatively or additionally, even if the properties of the meters change, if the change is known and coverage is supported, the reconstruction method can be adjusted (for example, places and / or amplitudes in the space of triangles) to take into account environmental influences.

Another possible environmental variable is the type of solvent used, as some meters may not be highly soluble in water, so measurements may use non-standard solvents.

In another example, the meter may exhibit conformational changes that are desirable for measurements with small changes in the environment, such as the concentration of calcium ions. It may be desirable that the meters do not exhibit the same sensitivity as a target protein to changes.

Alternatively or additionally, meters can be designed or selected to vary under different environmental conditions, thus, for example, allowing a single target to take multiple measurements, each in different environments.

13.7 Uniqueness of meters and overlapping sides and triangles

As indicated above, two different lengths of the sides of the meter can correspond to a specific configuration of the site of interaction, for example, using a site of interaction capable of binding two different groups and / or due to the flexibility in the meters (and / or targets), which cannot be completely removed.

In an illustrative embodiment of the present invention, the overlap between the measurements of the meters is controlled to be substantially constant in the space of the meters. Alternatively or additionally, overlap is minimized. Alternatively, at least a minimal amount of overlap is encouraged, for example, to compensate for various accidents, if the meter does not communicate or the study is unsuccessful or to provide additional connection information.

It should be noted that even if a substantially rigid meter is used, the level of tolerance is inherent in the interaction so that there is always some degree of freedom, although it is possible with the expenditure of the binding force.

If the degree of overlap is known, its effects can be compensated by the above

- 45 010258 by means of reconstruction, for example, during clustering. Alternatively or additionally, if the expected degree of overlap does not exhibit the expected effects, the measurement is doubtful.

However, in an illustrative embodiment, the present invention provides a greater degree of overlap, for example, by a factor of two, three or more repetitions of triangles. Fragmentary overlapping can be provided, for example, by using groups that do not have orthogonal affinity (in the detectable range) and / or as a result of partial overlapping between triangles. However, usually the exact same triangle cannot be repeated, for example, due to the difference between the frames and / or other groups and / or the effects of other groups within the frames.

Thus, alternatively or additionally, for random overlapping, some or all of the triangles are repeated between the meters. In an illustrative embodiment of the present invention, such a repetition is used to compensate for the effect of spatial inconsistencies and / or other unexpected chemical behavior exhibited by some meters. Alternatively or additionally, repetition is provided to facilitate determination of which triangle is connected based on the binding of the meters. For such an effect, the meters can be selected so that the overlap is less between the meters relative to other triangles, which include two meters. However, it is obvious that if the frames are quite different, the probability of overlapping most triangles in one frame with most triangles in another frame is small. This can help distinguish overlap between different wireframes and meters. Alternatively, similar frameworks can be used in such a way that a greater degree of overlap of the triangles of the same meters is achieved. It should be noted that part of the overlap is provided using a coefficient at which the meters can have some degree of flexibility, therefore, the same array of triangles of tie points can be compared using triangles of different sizes. In an illustrative embodiment of the present invention, a library is designed such that an array of points of each triangle can be matched to match with at least one larger triangle and at least one smaller triangle. Such overlapping may be optional or instead of overlapping a type of repetition in which substantially the same triangles are provided at least twice.

Optionally, the order of the groups in a particular framework is adjusted to take into account the expected spatial mismatches, for example, to ensure that at least some triangles will not have the same spatial mismatch problems, like other triangles.

Alternatively or additionally, a mixture of meters having the same triangles but different spatial mismatches can be heterogeneous in the same array to help avoid the problem of spatial mismatch.

In an illustrative embodiment of the present invention, although the overlapping of the triangle is usually not the same, the triangles of the gauges of at least some of the library, for example 20, 40, 60%, or any smaller, intermediate or larger percentage are selected so that the distribution of triangles in the space of triangles forms a relatively discrete grid with clusters of triangles near the grid points. Alternatively, at least some of the libraries, for example 20, 40, 60%, or any smaller, intermediate or higher percentage, are selected so that the overlap of the space of the triangles is relatively uniform with little clustering. As noted above, overlapping may be suitable to overcome various causes of non-binding. However, a larger overlap may mean a larger library.

It should be noted that the degree of overlap does not have to be uniform. For example, some sizes of triangles may be more susceptible to spatial inconsistencies (for example, if they all use large frames), in which a case of larger overlap may be provided. Optionally, clustering methods take into account the degree of overlap, for example, to determine the threshold when deciding whether a triangle is connected.

13.8 Weight and size of the meter

In an illustrative embodiment of the present invention, the meters are selected so that they have a minimum mass. With increasing mass, it is believed that the triangle becomes more energetic and less suitable for binding. Alternatively or additionally, a larger mass means a larger size and a greater chance of spatial inconsistencies. In an illustrative embodiment of the present invention, the frames are selected so that they have a mass of up to 200, not including groups. Perhaps the increased mass of the benzene ring groups is compensated, at least in part, by its increased affinity. Alternatively or additionally, the gauges are selected by size, for example, so that it is larger than 4 condensed rings (for example, about 10A). Alternatively or additionally, if molecules are selected for inclusion as a meter, the selection is unsuccessful if the molecule is large enough

- 46 010258 or massive enough. It should be noted that in some cases, consideration of size is relative. For example, in some embodiments of the present invention, it is desirable that the triangle has sides of the order of the size of the frame. Small triangles on a large frame may not be taken into account when considering distributed triangles with a specific meter, and you may need to provide them with smaller frames.

You should take into account that these examples are not limited and the meters can be larger and / or have a larger mass or be limited to be smaller and / or have a lower mass, for example, depending on the application or implementation.

14. Specific and general development of a set of meters

14.1 Example Coverage Library Size

According to some assumptions, the following is an estimate of the number of meters and triangles in the complete coverage library for small molecules on protein targets.

Assuming that the range of lengths to be coated is 9A, in increments of 1A, the number of possible triangles is 10 * 10 * 10 / (2 * 3) (coefficient 2 for a triangle with equality and coefficient 3 for a rotational degree of degeneracy). Admitting 10 groups and group directions, giving 166,000 triangles. Assuming an overlap factor of 3 and 5 triangles per meter, giving approximately 100,000 meters. Such numbers are only approximate in order, but can serve to clarify the following description of library development.

It may seem that the size of the library depends on the space of the triangles intended for coverage, the degree of accuracy, the complexity of the meters and the degree of overlap. Any of them may vary depending on illustrative embodiments, for example, creating libraries with a number of meters from 10,000 or more meters to 1,000,000 or more meters. An illustrative intermediate library size includes 30,000, 60,000, 80,000, 200,000 and 550,000 meters. Additionally, the library may include elements, not meters, or may form part of a significantly larger screening library, for example, as described above. In general, there are more meters in the library, more work to use them as a whole. However, greater accuracy, specificity, and coverage may be available as the size of the library grows.

An example of smaller meter libraries are those that have only 7 groups, reducing the sampling distance to 8 and / or reducing the overlap factor to 2. Libraries smaller and larger and / or other modifications to the library parameters may be provided in some embodiments of the present invention, as well as various incomplete libraries.

In another example, all meters are designed to include a single triangle (or a small number) in which a representative of a plurality of approximately 166,000 meters is needed (if there is no overlap). In such a library of specific meters, the initial stage of clustering is optionally skipped. However, it is noted that meters can typically include at least inadvertently more than one unit of measure, so that clustering may still be suitable. In some cases, a group is provided on the meter to prevent part of the meter frame from complicity as part of the unit of measurement, and / or to reduce the number of different triangles provided by the particular meter.

14.2 Selecting a subset of meters

A particular type of gauge library is a subset library, which may be smaller than the standard library (but it may be larger, for example, if it has a greater resolution of group lengths and / or types).

In an illustrative embodiment of the present invention, only a subset of all meters is used for a particular measurement. In some cases this happens because iterative approximation is used, which does not use all suitable meters at each stage. Alternatively or additionally, this may be desirable to reduce the number of studies performed. Alternatively or additionally, this may result from a large overlap between different meters. In an illustrative embodiment of the present invention, the meters are selected so that they work better in the environment (for example, used temperature, pH, solvent) and / or show some adverse interactions with the target and / or study, for example, in a cell study. Alternatively or additionally, this may be the result of failure to create a complete coverage library, for example, as shown in the example above, which can be almost universally used for all small drug protein meters.

It should be noted that one potential advantage of rigid meters is that the geometry of many rigid molecules is subject to minimal environmental changes, even if their chemical behavior is affected. This can allow a set of meters to be more versatile.

In an illustrative embodiment of the present invention, the meters for the subset are selected based on the type of target, for example, the expected range of distances between

- 47 010258 actions.

Alternatively or additionally, the meters are selected sensitive to the desired measurements. For example, if a certain interaction site has an unknown size, but it is known to have a weak affinity, a denser selection of the group size range for such an interaction site can be used (for example, for meters that are expected to associate with such a place).

Alternatively or additionally, the meters are selected sensitive to recognition of the available types of drugs, for example, the types of possible directions of hydrogen bonds in drugs. Alternatively or additionally, meters are selected to better distinguish between two potential drug substances by providing a better resolution of the differences between drug substances.

In some embodiments of the present invention, the meters are selected so that an approximate, correct model can be reconstructed, even for those parts of the target that use lower resolution meters. Alternatively, the meters are selected to determine whether a particular meter should communicate with the target, so only the meters needed to measure a smaller range of possible configurations are needed.

Optionally, meters are selected for the desired type of binding match, for example, if it is known that the target and / or potential drug substance include sulfate bonds, meters including sulfate groups are used.

In an illustrative embodiment of the present invention, a method for selecting a subset of meters comprises:

(a) determining the application of a subset of meters;

(b) defining a rule or rules for selecting meters that satisfy the specified application (for example, sizes, groups, densities, etc., etc., as indicated above);

(c) selecting from the library a plurality of meters that satisfy the specified rule (s); and (s) optionally determining whether the resulting library is suitable for providing the desired information for the specified application. For example, modeling can be used to see if the test results are suitable for the result of the reconstruction (for example, based on the studied binding ratio, coating density, properties or target and / or the degree of overlap required to distinguish between triangles on the meter). In another example, the information is incomplete information, and the simulation speeds up viewing if the information can be different.

14.3 Development of a library of meters

Table I

Title ABOUT thiophene n ό 1H-pyrrole ύ furan ABOUT benzene ABOUT pyridine ABOUT pyrimidine 0 pyrazine bH-trieno [2,3-b] pyrrole OU 1, b-dihydropyrrolo [2,3-b] pyrrole with 1H-indole With trieno [2, Z-ά] pyrimidine

I 6,7-dihydropyrazolo [1,5-a] pyrimidine

- 48 010258

with quinoline with isoquinoline sf quinoxaline CE 3,4-dihydrobenzo [e] [1,4] diazepin-5-one V CO 3,8-dihydro-4H-pyrrolo [2,3-e] [1,4] diazepin-5-one ν _Ν <y 3,4-dihydrotrieno [2,3-e] [1,4] diazepin-5-one ν οϋ 3,6-dihydro-4H-pyrrolo [3,2-e] [1,4] diazepin-5-one ΰ 5H, 11H-dibenzo [b, £] [1, 5] diazocin-6,12-dione 1,4-dihydro-10H-1,4,10- ABOUT triazabenzo [a] cyclopenta [e] cyclooctene-5,11-dione 4H, 10H-1-thia-4.10- 0 diazabenzo [a] cyclopenta [e] cyclooctene-5,11-dione 0 dipirollo [1,2-s; 2 ', 1'-e] imidazol-5-one sg% 1,4,7,9-tetrahydro-1,4,6,9— ABOUT tetrazadicyclopenta [a, e] cyclooctene-5, 10-dione / V TX 4,7,9-trihydro-1-thia-4,6,9- on ABOUT triazadicyclopenta [a, e] cyclooctene-5,10-dione o ΓΪ g ³ 2,4,9-trihydro-1 lambda * 4 *, b-dithia-4,9- <gu / ^ ABOUT diazadicyclopenta [a, e] cyclooctene-5,10-dione 6.9-dihydro-5H-1-thia-5.8.9- triazacyclopenta [a] azulen-4-one bj 3,10-dihydro-4H- [1,4] diazepino [5,6-b] indol-5-one

- 49 010258

0 3,6-dihydro ~ 4H- [1,4] diazepino [6,5-b] indol-5-one 7,8-dihydro-1H-1,7,10-triazacyclohepta [e] indene 6th -g 8,9-dihydro-ZN-Z, 6,9-triazacyclohepta [e] indene 10-he b 7,8-dihydro-1Η — 1,5,8-triazacyclohepta [1] indene 9-he axis 8,9-dihydro-5,6,9,11- tetraazacyclohepta [b] naphthalene-10-one ooh 3,4-dihydro [1,4] diazepino [5, 6-b] quinolin-5 ~ he 8,9-dihydro-4,8,11-triazacyclohepta [a] naphthalene- 7th SOYA 11H-10,11-diazabenzo [b] fluorin οΆ s _r n a-hydroxy acids mA pss * a-amino acids r Oh copolymer gels l bicyclo [2.2.2] octane 2-methylene-2,3-dihydrobenzo [1,4] dioxin D) WH 6,7-dihydro-2H-pyrazino [1,2-a] pyramidine School 9H-fluorene BUT 1,4-diazabicyclo [2.2.2] octane

- 50 010258

Λ 1-azabicyclo [2.2.2] octane pyrrido [2,3-ά] pyrimidine ° Ό. 5-methylene-1,5-dihydropyrrol-2-one dYa and benzo [4,5] imidazo [1,2-a] pyrimidine υ 1,4-dihydrobenzo [4,5] imidaeo [1,2-a] pyrimidine Lee Μ 4, Yu-dihydro-1,4a, 10-triazapenantren-9-one $ 1,5-dihydroimidazo [1,2-a] pyrimidin-2-one 1,2,3,5-tetrahydroimidazo [1,2-a] pyrimidine thiazolo [3,2-a] thieno [2,3-6] pyrimidin-5-one X? 1,9-dithia-4a, 1O-diazacyclopenta [b] fluoren-4-one 5,6-dihydro-1-thia-5,7,8,9atetraazacyclopenta [e] azulen-4-one ά \ 6,10-dihydro-5H-1-thia-5,7,10- triazabenzo [e] azulen-4-one τΡ ζΐ 4,5-dihydro-3-thia-4,5a, 10 triazacyclopenta [a] fluorin ssya 8H-1-thia1diclopenta [a] indene dr $ 3-thia-4,5a, 1O-triazacyclopenta [a] fluorin 6,7,9,11-tetrahydro-10-thia-b, 9-diazaindeno [1,2- a] azulene-5.8-dione

- 51 010258

units 2,3,6,7,12a hexahydropyrazino [1 ', 2': 1,6] pyrido [3,4- (Lg ⁰ B] indole-1,4-dione αία 5,10-dihydro-4H-2, Za, 10 triazacyclopenta [a] fluorin oR 5H-pyrido [4.3-b] indole Sed IN-indolisino [1,2-b] quinolin-9-one s & g 1,2-dihydro-2,4a, 9, triazanthracene-3,10-dione gray bH-isoindolo [2,1-a] indole 1,5-dihydrobenzo [b] [1,4] diazepin-2-one 5,10-dihydrodibenzo [b, e] [1,4] diazepin-11-one 5,11-dihydrobenzo [e] pyrido [3,2-b] [1,4] diazepine 6th 0-Ζ 4,9-dihydro-3-thia-4,9-diazabenzo [ί] azulen-10-one units benzo [d] quinoxaline units pyrazino [2,3-b] quinoxaline about pyrido [2,1-b] quinazolin-11-one ¢¢ 0 1-thia-4a, 9-diazacyclopenta [b] naphthalen-4-one units 2-methylene-4H-benzo [1,4] thiazin-3-one

In an illustrative embodiment of the present invention, the groups are Me (methyl), Ε1 (ethyl), Pr (propyl), PE (phenol) CO ₂ H, OH and ΝΗ ₂ . Although groups can be connected to any of the K. sites, not all possible meters are necessary, as explained above. The indolysin framework can have either COOH or ΝΗ ₂ in K1, both of which are given in the table. More specifically, it was found that in the general case, with four or five points of attachment, it is possible to cover the entire range of triangles with M groups using only about M ³ different meters. This is assumed to be generally true (for example, the exponent is not much larger than 3) for frames with a higher number of attachment points.

It should be noted that even if the library does not cover all possible triangles, viable reconstruction is still possible for many drug targets and / or a significantly convenient model associated with the library. Also, as noted above, in some cases, incomplete reconstruction is suitable. Also, as indicated above, the correspondence of the gauges may be applicable as leaders and / or for rejecting leaders, even if reconstruction is not possible in some cases. In some embodiments, implementation of the present invention, the failure of the method is usually self-evident and does not create an unproductive search for non-existent leaders.

Alternatively, to construct a library of gauges from labels, at least a portion of the library can be created by scanning existing libraries for molecules that include triangles having desired sizes and / or groups. Optionally, molecules that are small and stiff are selected as described above. This type of library, for example, may not be based on a set of wireframes.

14.4 Method of building libraries

From the above, it should be understood that there are many methods that can be used to construct the library. The following illustrative methods are described, at least

- 52 010258 least, in part to illustrate various applications of the above rules:

(a) library parameters are selected: for example, the range of coverage and accuracy desired for the library;

(b) selecting groups for the library;

(c) selecting a framework;

(b) create gauges from the frame;

(e) add the created gauges, if appropriate;

(ί) repeating steps (c) - (e) until the library covers the range with the desired accuracy and / or coverage; and (e) optionally, testing the library.

According to an illustrative embodiment of the present invention, a resource allocation algorithm is used, for example, a greedy method or a first approximation method. Such names refer to how to select from a set of possible resources, a resource that is placed at a specific time, for example, which meter to choose for a library from the available meters on a frame or which frame to add to the library. Many such methods are known in the art and can be used, noting that in some embodiments, the implementation of this method is not required to provide an optimal solution, correct operation, or informed decision.

An alternative way is to design a library based on selection. In this method, existing libraries of molecules are scanned for molecules that have properties suitable for the meter (for example, as described herein). The resulting potential meters can be filtered to remove excess. However, it is expected that in the current state of public libraries, scanning of such libraries will not result in a complete library of meters. Optionally, such a selected meter library may be complete using other techniques, such as constructing meters based on a framework.

It should be borne in mind that for a given large number of possible meters and a smaller actual required quantity, there are many optimization methods for selecting a suitable and / or optimal set of meters that satisfies the required quantity. As noted above, the selection may be based on an application in which a library is determined and / or based on consideration, for example, of density, chemical behavior and synthesis ability. Additionally, part of the library can be replaced, for example, by a set of meters constructed from other frameworks, or by using molecules selected from a library of potential leaders. In an illustrative embodiment of the present invention, in step (e), the designed library is optimized, for example, by removing excess and ensuring that the desired distribution (e.g., triangles, chemical properties) and overlap (e.g., lengths and / or groups) are compliant and / or are optimal.

14.5 Wireframe selection method

In an illustrative embodiment of the present invention, the carcasses are usually selected so that they have some desired properties, for example, one or more of the following:

(a) small size;

(b) stiffness;

(c) suitability for combinatorial chemistry;

(b) the inclusion of a set of points of attachment, for example, 3, 4, 6, 10, 12, or any smaller, intermediate or greater number, for joining groups and / or chemical markers (for example, for binding analysis, chemical manipulation);

(e) the geometric arrangement of the points of attachment so that a range of sides of the triangle can be provided;

(ί) 3Ό structure, for example, flat or three-dimensional may be preferable for various situations;

(e) the number of excess protruding parts (in some cases it may be desirable to have few), to which groups may or may not join so that the excess corresponds to a complete framework on which it is suitable (for example, for a library or for specific triangles) groups determine the shape of the frame; and (11) solubility (can be determined, for example, based on the number of polar atoms in the framework).

In general, when providing more attachment points for groups, the wireframe is more able to provide triangles of different sizes, however, this can adversely affect the wireframe (and the size of the meter), and many triangles can be useless. In the general case, in the framework it may be useful to designate only some potential attachment points as points that are intended to be used. This can reduce the number of different synthesis methods and / or contribute to their uniformity.

Not all or even any of these properties are essential in some embodiments of the present invention. As a practical material, small rings and ring chains

- 53 010258 pi satisfy these criteria. Thus, in an illustrative embodiment of the present invention, a set of scaffolds can be created by reviewing existing known rings and small chains for molecules that satisfy the desired criteria. In an illustrative embodiment of the present invention, during this type of selection, forces are applied to the selection of frames having a range of sizes (e.g., distances between attachment points) so that a range of triangles can be created using frames.

In addition to the framework criterion, in general, the choice of frameworks for a library can impose other criteria, for example, which frameworks create a library for measuring coverage and / or a range of chemicals and / or require a relatively small number of methods of relatively small complexity to create meters.

In an illustrative embodiment of the present invention, a method for selecting a meter is as follows. For this part of the existing library, from the list of suitable potential frameworks, a new framework is selected if it meets at least one of the following criteria:

(a) the framework creates a large number of triangles that are missing from libraries, for example, 10, 50, 100, or any smaller, intermediate, or larger number, such as the number in a user set;

(b) the frame creates at least one (or a small number of triangles, such as less than 20, less than 10, or less than 50 or any other user set size) a triangle that bypasses the creation using other frames and forms the missing parts Libraries

(c) the framework has a significant amount of known chemicals (for example, methods for manipulating and / or adding groups); and (b) the framework adds the opportunity for the desired amount of overlap.

In general, if you create more meters, you can easily replenish the library. However, not all frames can create large numbers of suitable triangles.

It should be noted that in some methods of developing libraries based on discrepancies, each element of the library is selected so that it differs as much as possible so that this type of selection methods and / or at least some of the criteria used cannot be applicable and run into traditional ideas.

It should be noted that as soon as the library is full, consideration of criterion (b) can become more significant with the possibility of searching or constructing a frame that has the desired properties (for example, to form the required triangles). Moreover, the search can lead to the choice of less rigid frames, for example, to guarantee coverage, or due to the lack of suitable more rigid frames.

In an illustrative embodiment of the present invention, during the optimal library optimization step, the frames are evaluated for their quality (for example, meeting the criteria of the meter), the number of triangles being created, and / or the uniqueness of the triangles being created. The framework can be removed from the library if it is determined that it is less suitable or is not necessary, based on one or more of these considerations.

One difference between the frames is the number of rings in the frame. In the general case, as the number of rings increases, the size and weight of the frame are therefore valid. For some applications, the number of rings in a crucian can be used as a heuristic to determine which approximate sizes of triangles and wireframes can be provided. For some applications, multi-ring frames may be necessary. Alternatively or additionally, a triangle with one or two rings can be used for a small triangle and / or to reduce spatial inconsistencies.

14.6 Meter selection method

In an illustrative embodiment of the present invention, the meters are generally selected so that they have some desired properties, for example, one or more of the following:

(a) small size;

(b) a large number of triangles;

(c) a high or other desired binding affinity, for example, in the range of 1-100 micromoles;

(b) rigidity;

(e) attached groups that define the volume of the molecule;

(ί) relatively uniform binding ability for all groups, for example, a coefficient of ten between groups and a coefficient of 100 between molecules in the library, however, in other embodiments, other, smaller or larger coefficients (for example, ~ 1, 5, 20, 50, 130, 250, 1000, or any smaller, intermediate, or larger ratio) may be provided for one or two criteria; and / or (e) chemical behavior, such as (ί) solubility, for example, in a natural solution of the target (or approximately that), for example, pH adjusted water with some detergents, such as

- 54 010258

DMSO to promote solubility, (ίί) lack of reaction with expected contaminants, (w) lack of chemical reaction (criterion of covalent bonds) with target proteins, i.e., with amino acids or known conventional combinations of them and / or with a substrate, (ίν) desired behavior over a range of properties.

In general, higher binding order information means that the studies have the same meaning. However, it is usually impractical to provide such narrowly defined substances, and some freedom in providing a real set of chemicals is useful.

When creating a library (or part thereof) by selecting meters from existing libraries for screening molecules, each molecule is screened, for example, for the desired criterion. The molecule may be selected or rejected. Alternatively or additionally, the molecule may have an assessment of the suitability associated with it. Similarly, a set of potential meters can be created from frames.

In an illustrative embodiment of the present invention, the meters are selected from the created / selected set based on one or two devices (for example, relative or absolute) and to satisfy a group of criteria. In an illustrative embodiment of the present invention apply one or more of the following groups of criteria, for example, as a binary criterion or as part of an assessment:

(a) Such uniformity of triangles provided and / or their correspondence to missing triangles.

(b) Matching the flexibility of the meters and / or individual triangles with the desired flexibility.

(c) The shape of the meter as a whole, for example, elongated or rounded. A shape can be considered, for example, when building a library in which the shapes are modified so that spatial inconsistencies could not deflect all of the defined triangles. For this purpose, the shape of the meter can interact with the location of a certain triangle, for example, if the same triangle is found on two elongated meters, it may be desirable that the triangle is in the axial direction on one of the meters and in the trans-axial direction on the other. Alternatively or additionally, consideration of the shape refers to the three-dimensional shape of the meter and / or the relative arrangement of the triangles in the meter.

(6) That some non-triangular units of measure are found, for example, specific non-triangular units of measure are provided or such a uniform (or other) distribution of such 4-5 or other multipoint units of measure.

It should be noted that for meters and / or frames, device definitions, for example, one or more simulations of use and molecular analysis software, testing a chemical library and / or literature search for the same or similar chemical compound, may be included.

The above selection method can be used in the development of one universal library (or a set of such libraries for widespread use). However, it should be noted that some, similar or other selection methods can be used to create personal and / or special libraries, search for meters or units of measure for specific properties, and / or to determine a meter and / or to create a framework.

14.7 Meter Synthesis

In some embodiments, implementation of the present invention, the creation of a library of meters from frames can help in serial synthesis of meters. In libraries that are not (or almost none) based on frameworks, standard synthesis methods can be used.

In an illustrative embodiment of the present invention, meters are synthesized, for example, using liquid phase methods, as described below, and impurities are removed using standard methods, for example, using HPLC.

In an illustrative embodiment of the present invention, a parallel synthesis method is used in which a set of meters is synthesized once and then isolated. It should be noted that in some embodiments of the present invention, only a small number of meters that can be created using the frame is really necessary. Alternatively or additionally, even if specific meters cannot be created, a sufficient number of alternative meters may be suitable to provide coverage and / or overlap of the desired space of the triangles. For example, on a 5-point frame with 10 groups, 10,000 combinations are possible, of which 1000 is enough to cover the space of triangles. Thus, the choice can be, for example, special, such as on the basis of an actual result (for example, a relative result) or on the basis of preliminary development of the library.

In an illustrative embodiment of the present invention, combinatorial chemistry methods are used to attach the groups, each to the point of attachment of the framework, optionally so that all combinations of groups are created. Each final compound is created by attachment to a polymer matrix (for example) for easy separation. Matrices can be color coded for

- 55 010258 relief in the separation and / or identification of the created meter.

Alternatively, other solid phase methods are used, for example, as described below or as is known in the art.

14.8 Developing a Mixed Library

As noted above, a complete universal library is not required to be suitable. In addition, a meter library can be included in a standard screening library. In an illustrative embodiment of the present invention, at least 0.05, 0.1, 0.5, 1%, 5, 10, 20, 40%, or any smaller, intermediate, or larger percentage of molecules in a library used for screening, measuring, and / or other applications, contains molecules similar to meters. Of these meters, for example, less than 50%, or more than 30, 60, 80, 90%, or any smaller, intermediate or greater percentage of meters are frame based meters in which the frame is used to create at least 5 meters with less than 20% overlap in triangles defined by joining groups. As noted above, since the library may include standard screening parts that provide a significant number of molecules, like meters, this can help when using the methods described in this description.

In an illustrative embodiment of the present invention, the library contains at least 5000, 10000, 20,000, 50,000, 80,000, 100,000, or any intermediate or more meters. Such meters can cover, for example, 5, 20, 40, 80, 100% or any smaller, intermediate or greater percentage of the space of the triangles, for example, with an overlap of 1.1, 1.5, 2.3 or any smaller, intermediate or greater degree. As noted above, when coverage is better, the degree of success may be higher, although at the cost of using a large library. Smaller libraries may be easier to use and still provide useful results in many cases.

One of the significant differences between gauge libraries and other leader libraries (e.g., diversity-based libraries) according to some illustrative embodiments of the present invention is that it is expected to use a relatively large number of gauge-based library matches. For example, at least 0.01, 0.05, 0.1, 0.2, 0.5, 1, 3, 5, 10%, or any smaller, intermediate, or greater percentage of the amount is expected to be bound. The percentage of binding may depend, for example, on the ratio between meters and leaders not meters in the library.

It should be noted that such percentages are not a simple amount. Rather, they are a quantitative difference from libraries, which more often than less does not lead to linking. With a greater likelihood of finding one or more leaders and more leaders, it is more likely that a drug substance can be found. However, with overly probable binding, the quality of the information provided by the binding decreases.

A library may also include a mixture of three point units and higher valency units. Although any meter that includes more than three groups contains units of higher valency, in an illustrative embodiment of the present invention, a library is designed to cover a higher valency space. For example, a library spans at least 0.1, 0.3, 0.5%, or at least 1%, or any smaller, intermediate, or greater percentage of space with units of higher valency. Coverage can be, for example, continuous (for example, the entire library with a lower resolution or part of a library with a higher resolution), or it can be discrete (for example, isolated parts of the library). In general, units of higher valency may require a very large number, for example, 20,000,000, for the equivalent coverage for a 100,000 library of triangles, so commercial implementation may depend on the effectiveness of even larger parallel studies than are available today. Units of higher valency are optionally provided for greater flexibility so that lower resolution is required to cover the space.

14.9 Ensuring library reliability

In an exemplary embodiment of the present invention, a library is constructed once and / or various quality assurance processes can be performed during its construction. In one example, a library is analyzed to ensure that it satisfies the coverage, overlap, and / or exact set of criteria for the library. Any missing triangle can be provided at this point or marked as missing. Alternatively or additionally, molecules with low solubility or high toxicity are removed and / or replaced with molecules exhibiting similar spatial chemical configurations.

In an illustrative embodiment of the present invention, feedback from the use of the library is used to calibrate the library, the reconstruction process, and / or to assist in the development of the library.

In an illustrative embodiment of the present invention, a theoretical bib model

- 56 010258 libraries are compared with its actual behavior, for example, ongoing testing studies against randomly selected targets having a known and / or unknown structure. Two examples of molecules with known structures fully reflect proteins and structures constructed from DNA or RNA, with optionally attached elements. Optionally, the targets are nonrandom, and are selected from the assumption of some testing in the theoretical model of the library. Alternatively or additionally, calibration is provided by analyzing the results of actual library applications each time.

In an illustrative embodiment of the present invention, one or more of the following data is provided using such an analysis:

(a) investigation of binding ratios for meters and families (eg, similar) of meters;

(b) the relationship between environmental conditions and the ratios of binding and / or conformational changes for one or more meters;

(c) Bayesian probability of spatial inconsistencies between meters (and their triangles) with overlapping triangles;

(6) the actual degree of overlap between the triangles;

(e) the relationship between the type of triangle and the binding of the meter; and / or (1) parameter values (e.g., thresholds) for various algorithms.

Other properties of the library, for example, the general rigidity of the meters and the correctness of the values in the data bank can also be provided using such an analysis.

In an illustrative embodiment of the present invention, as a result of the above conclusions, the library is improved, for example, by removing duplicate meters and / or by searching for meters to create the missing triangles.

Alternatively or additionally, as a result of the above conclusions, the creation of libraries and sub-libraries is further modified to take into account calibration information, for example, with respect to a specific method related to specific meters and / or, in relation to a general method related to a statistical deviation of the behavior of frames / or families of meters from their suitable theoretical models and / or as parameters of such models.

Alternatively or additionally, the reconstruction process is calibrated, for example, to better determine which triangle corresponds, the actual coverage of each triangle, the spatial shape (in the space of the triangles) of the correspondence and / or the relative binding strength of the various units of measurement of the triangles and / or meters.

14.10 The human factor during library development

The library development method can be automatic, semi-automatic, and manual. In general, if a plurality of potential meters and / or frames are available, and suitable modeling software is also available, automated development may be provided. One example of this is that when a complete library is available, the selection of a subset can be fully automatic if the desired parameters are specified. Some libraries can be created automatically, in any case, for example, selecting meters from existing libraries and / or selecting frameworks from existing libraries. It may be necessary to determine the ease of synthesis, which is done manually if earlier information is not available. However, it is noted that in an illustrative embodiment of the present invention, the scaffolds are selected so as to define known chemical behavior and synthesis paths so that the addition of groups would require little or no research work. However, in some cases, a person may need not only a choice between alternatives, but really a search for a specific missing meter or a proposal for developing a frame. However, it is noted that the mathematical description of the library according to some embodiments of the present invention helps and may allow full or almost complete automatic creation of the library using constructive synthesis and / or analysis of existing molecules. Perhaps such a library can then be optimized, for example, as described above, perhaps manually, especially to help provide a library that is easy to synthesize.

As noted above, the reconstruction process may be fully automatic or it may include a manual approach. However, in the general case, it is believed that a high ratio of meter binding hits can reduce or eliminate any need for human intervention, at least at some stages of the drug search. Of course, after the completion of the mapping, the user, the person, may wish to test the influence of various assumptions, for example, how the reconstructed layout depends on various assumptions on the correspondence of the target. Also, in some cases, an expert, a person (or an expert system) can be used to make choices among alternatives or choose suitable leaders, since in many cases the method can create a small number of possibilities, from which one or two should be selected, thereby leveling out the possible high cost.

In an illustrative embodiment of the present invention, one of the points of application

- 57 010258 human intervention in the method of searching for a medicinal substance is the development of candidates for medicinal substances that correspond to the final pharmacophore (for example, models). It has been noted that various software exists to assist or automate this step. However, usually (at present) in assessing the feasibility of synthesis for complex molecules, a person’s opinion is better. If the proposed medicinal substances are developed by combining meters or simple fragments, however, automatic evaluation and, possibly, methods of creation may be acceptable.

15. Experiments and examples

15.1 Experiment 1

One of the measurement methods described above was tested using the following experiment.

In this experiment, known H1U-1 protease inhibitors are analyzed to find a set of triangular units of measure that could find binding to the H1U-1 protease. A set of molecules, including triangular units of measurement, is selected, physically analyzed and it has been shown that they have the expected binding to the NSU-1 protease. The results indicate that triangles are viable geometric substructures that can be used to measure targets by binding.

The following substances in the BDB (Protein Database) were isolated as H1U-1 protease structures with known related inhibitors: 1a) t 1a) x 1άίΓ 1dpo 111bt 1 11u5 11ψ5 ΙΗρν 1yr 1115d 1111e ΙΗΐΓ Shchd 11ινί 1Ην_ί 11ιν1 <1ΗνЬ 2bρν 2bp \ x 2bpx 2bp 2bpr 2ir_) 311H 51ινρ 7ir_).

The structures are superimposed using the protein as a reference system so as to superimpose the spatial position and orientation of the inhibitors. Then the molecules of the inhibitors are divided into groups and groups are grouped in space. Strong binding sites are identified based on the same group in different molecules that binds essentially to the same binding site in the protease. Confidence in such places is increased by verification, and the protein groups in such places were compatible with the groups of the inhibitor molecule.

The triplets of the inhibitor groups at the sites of strong binding are selected as triangles. It is expected that the meters, for example, a set of meters, as described above, which has such triplets, are connected, or at least some of them could be connected.

Triplets are used as query input data for a search in ME LSE-8C (an accessible chemical directory for screening). Molecules that match the requirements (groups and size) and stiffness requirements are selected as shown in the following table.

- 58 010258

Table II

No. Compound mm molecular weight) Density (g / ml) Catalog number mg per 1 mm in 10 ml one 276.35 5-83425-4 2.8 2 403.26 1.006 36,667-6 4.00μ1 3 391.35 3-63995-8 3.9 4 40832 3-84651-1 4.1 5 BUT 324.55 8-2210-2 3.2 6 464.56 J-15419-9 4.6 7 445.57 5-22759-5 1.9 8 0¾ ³ 445.57 8-22675-0 1.9 nine oyao 446.17 5-95285-0 4.5 10 412.53 5-9757-9 4.1 eleven 464.56 K-15449-0 4.6

- 59 010258

12 F 438.57 P-15358-3 4.4 thirteen 436.55 K-15353-2 4.4 14 422.53 K-33994-6 4.2 fifteen 204.16 8-52812-9 2.0 sixteen 298.34 5-6426-3 3.0 17 y- 200.32 0.687 46443-0 2.26μΙ eighteen cf ^ he 234.34 27302-3 2.3 nineteen 280.37 H-22433-2 2.8 twenty lk not 236.36 44642-4 2.4 21 R X> O 0 268.33 8-4228-6 2.7

- 60 010258

22 344.38 ΝΡΒ-0140Ϊ 3.4 23 377.51 rice 03605 3.8 24 it 0¼ 245.37 3ΡΌ-03358 2.5 25 y ^he st ^ xT 350.31 rice-03637 3.5 26 408.63 KZS-03257 4.1 27 about. O ^ nA ^ O ό 435.49 3143-01334 4.4 28 about 460.32 Gis-02058 4.6 29th 456.59 rice-02951 4.6 thirty 477.41 VTV-14801 4.8 31 about 280.29 VTV-11623 2.8

- 61 010258

32 P-A ¹ P ^ 255.43 ΚϋΟ-03631 3.0 33 212.25 UR-00720 2.1

34 BUT 0M4 302.41 85,612-6 35 sfu ° ⁿ o 252.23 25,272-7 36 Αχο cHF 267.54 29,126-9 37 / E-- E 258.12 23,319-6 38 oi ^Βγ 4 | Αζ ³ 'CH. 265.94 30,118-3 39 303.34 16,263-9

As expected, molecules having numbers up to 33 exhibit binding behavior due to the fact that they include at least one triplet. Molecules numbered 34 onwards are similar in appearance but do not include the required triangles.

All molecules were indeed analyzed and provided to show activity (effect on the Ηΐν-1 protease) at various concentrations (between 10 and 1000 micromoles). From such molecules, it was found that the molecules of compounds 1-33, about 60%, are active, more specifically, molecules 7, 9, 23 and 27. Molecules 34-39 were also analyzed, which, as expected, showed no activity.

As noted above, such results have shown that typically meters that have a triangle as the unit of measurement that correspond to the layout of the target should often communicate in a detectable manner.

15.2 Experiment 2

In this experiment, the results of studies performed by others for reconstruction use the spatial layout of the binding sites of known molecules and then compare with the current state of the art.

N0 maintains a database of molecules that have been tested positively for кν activity. 43,000 results (published October 1999) are available at 1ι11ρ: //6ΐρ.ηα.ηί1ι.§ον. as public information, further results from ΛΙΌδ antiviral screening. Of these molecules, a subset is selected that shows at least an average level of activity and is stiff enough to allow the determination of the spatial position of all such groups. This results in fewer than 200 molecules. Triangles of groups are grouped in these selected molecules.

The grouping results show good agreement with the results of experiment 1, and triangles of molecules were found in BDB structures.

These results are presented to show that at least part of a suitable library

- 62 010258 can be created by selecting suitable meters from the available libraries faster than by constructing using wireframes. You should take into account that it may be necessary to determine the spatial positions of all groups, for example, only groups with high binding affinity. In some cases, low affinity groups may be removed.

16. The book of synthesis

The following is a synthesis book organized in chapters for some frameworks (and meters derived from them) shown in Table. I. The most important aspect of such a synthesis is to illustrate that suitable frameworks and meters are available and can be created using known chemical methods applied to standard or modified sources, and / or by changing their parameters in the expected manner. All of the contents of this book are hereby incorporated by reference. In any case, the incomplete library described in the appendix has at least a property that can serve in many cases to provide partial reconstruction and / or a significant increase in accordance with the leader.

It should be borne in mind that the new substances described in this book, methods for manipulating them, methods for their synthesis and groups of molecules are included in the scope of at least some aspects of the present invention, for example, a library including one, two, four, six, eight or any intermediate number of frameworks as described herein. Alternatively or additionally, the library according to an illustrative embodiment of the present invention includes at least 100, 300, 500, 1000, 2000, 4000, 10000, 20000, or any fewer, intermediate or more meters from this book. Although it is useful to select meters from this book, for example, by using the frames described in the present description, this is not required to cover part of the library.

16.1 Framework of the 6-membered ring of benzenes, pyrimidines

The synthesis of Biginelli dihydropyrimidine (the scheme is shown below) is a promising multicomponent condensation, which includes the single-stage cyclocondensation of β-ketoesters 2, aldehydes 3 and urea 4, which provide heterocycle 1, which can be oxidized to the corresponding pyrimidine group.

Some protocols are designed for ¹ Biginelli reactions in solution. However, in order to bring the reaction to completion, an excess of two or three components 2-4 is usually used, and purification steps are required. Solid-phase synthesis provides the desired dihydropyrimidines with good yield and excellent purity immediately after cleavage from resin ² (the scheme is shown below):

Recently ³ other methods for the solid-state synthesis of highly substituted pyrimidines have been published. In this work, the synthesis begins with a thiourea salt 5 bound to a resin, which is subjected to cyclocondensation with acetylene ketones 6 to form carboxypyrimidines 7 (the scheme is shown below).

- 63 010258

Tetrazubstituted pyrimidines a can be obtained using modified synthesis

First, imidine functional groups are formed on an acid labile resin to create a resin immobilized by imidines 23 ⁴ , urea 24 and guanidine 25 ² . Indeed, such amidines serve as Biginelli's first building block. The subsequent addition of two other Biginelli building blocks, namely 2 and 3, to 23-25 leads to the creation of dihydropyrimidine scaffolds 20, 21 and 22 ⁵ , respectively. Subsequent reduction of the ketone groups SchaBH4, WB30eB) leads to 14, 15 and 16, which after cleavage (TFA, ESM, 1: 1) followed by mild oxidation (ΟΑΝ, CH3SC) gave the desired pyrimidines 8, 9, ^5, and 10, respectively. CΑN can be removed after completion of oxidation by solid phase extraction (8ΡΕ) or using a simple 96-well 8eRask. Other oxidation reagents, such as Mn02 ⁶ , 0-chloroanil7, Ktp04 ⁸ and Cr03, AcOH, H ₂ 80 ₄ ^9, can also be used. In the case of H3 = 0Me (when β-keto ester is the building block 2), dihydropyrimidines 20, 21, and 22 are subjected to ester hydrolysis (L0H, THF, or a 5% alcohol solution of KOH ¹⁰ ) to obtain 4-carboxyhydropyrimidines 17, 18, and 19, respectively. Following the same procedure as for 8, 9, and 10 (1. TFU, ESM, 1: 1; 2. CA ^ CH ₃ SC), 17, 18, and 19 were subjected to interaction to create a sub-library of 4-carboxypyrimidines 11, 12, and 13 , respectively. It should be noted that in the case of asymmetric 1,3 diketones 2, a mixture of two isomers is obtained.

- 64 010258

Basic approach to tetrasubstituted pyrimidines

It was shown ¹¹ that hydropyrimidine-5-carboxylic acid can be converted to carbonyl azide, which in turn undergoes Curtius rearrangement to give isocyanate. This reaction gives an excess of 5-aminodihydropyrimidines A.

Pyrimidines may be unsaturated ketones. Recently, scientists published work ¹² on synthesis, which was obtained by cyclocondensation of amidines with α, β-65 010258 and describes the use of the Wittig reaction in the formation of α, β-unsaturated ketones at 8P for the synthesis of various heterocycles. This application proposes an alternative three-step synthesis of a pyrimidines a in solution, based on the formation of building blocks 26 α, β-unsaturated ketone as a key step ^12b-b , described below:

α, β-unsaturated ketones 26 can be obtained in good yield and purity by the Wittig reaction with a suitable aldehyde and the corresponding triphenylphosphonium bromide 27 with ΝηΘΕί under reflux in ΌΜΆ. The yield of phosphorus compound 27 is readily available from α-bromoketones 28 by the Arbuzov reaction followed by treatment with a strong base such as ΝηΘΕΐ. The reaction of ketones 26 with various amidines 23 ^12b-6 (Fig. 4) provides the desired tetrasubstituted pyrimidine sub-library a.

Small b-d sub-libraries having one or more constant functional groups on a six-membered aromatic ring are characterized by better solubility.

A series of 2,5,6-trisubstituted-4-oxo-dihydropyrimidines 29 can be obtained using 8P using a cyclization-cleavage strategy ¹³ from readily available amidines 23 and α, β, unsaturated carboxylic acids 30 ¹⁴ attached to the resin (see diagram below). Compound 30 is prepared by attaching a resin to acyl chloride 31 (modified from commercially available α, β-unsaturated carboxylic acids).

Compounds 29 ^13b can be oxidized (SAU CH ₃ SC) to the corresponding pyrimidines b. The solid-phase method for producing Knevenagel condensation products from a resin bound by malonates and malonic acids has the ability to produce hetero- and carbocyclic compounds (see diagram below).

The mono-ester of lactic acid (see chart above) is obtained from the macroporous resin Wang (AdgoRoge, Agdopai! Teplodomég) ^{15 by} treatment with Meldrum acids. The conversion of asymmetric ester 34 is achieved by treatment with trifluoroethanol and E1C followed by condensation of Knevenagel with aldehyde in the presence of piperidine acetate to obtain substituted methylene malonate 33. To obtain bulk resin 33 (2-10 g of resin), Knevenagel is condensed with a DeanStark trap to remove water equally increased the yield and accelerated the reaction. Malonates 33

- 66 010258 is treated with 10 equivalents of amidine 23 hydrochloride in a solution of dimethylacetamide (ΌΜΆ) with an excess of K ₂ CO ₃ to neutralize the HC1 salt of amidine at 70 ° C for 4-8 hours to obtain dihydropyrimidines associated with the resin 32. The progress of consumption of reagents can be monitored at ΕΤΙΒ spectrometer monitoring the adsorption of groups C = N and C = O. Oxidation of 23 with 0.2 M ammonium cerium nitrate (CAC) at ΌΜΆ ¹⁶ yields hydroxypyrimidines bound to the resin. Cleavage under acidic conditions (TFU / OSM, 1: 1, CT, 1-2 h) gives a second sub-library c (sub-library c exists in the tautomeric form of 4-pyrimidone).

The following are examples of the synthesis of mixed 6-membered rings with desired properties.

Amidines 23-25 are reacted in solution with commercially available [bis (methylthio) methylidene] malononitrile 35 (see chart below) in the presence of ΌΙΕΑ ¹⁷ to obtain the corresponding methylthiopyrimidines. Then oxidized with 1.2 equiv. t-CPBA in an ESM or Н2О2 ¹⁸ for the formation of intermediate thionyl derivatives 36, which are substituted by amine ΝΗ3 ¹⁹ (dioxane, room temperature), resulting after hydrolysis of nitrile (ΤΡΡΆ) ¹⁰ to the final aminopyrimidines 37. If OH is used instead of ΝΗ ₃ , then after nitrile receive the corresponding hydroxypyrimidines 38 ²¹ .

A series of various 3,4,5-trisubstituted phenols 39 can be synthesized in high yield using the cyclization-cleavage approach ²² .

- 67 010258

The main catalyzed reactions between α, β-unsaturated ketones and resin-associated acetonyl groups 42 (see diagram above) are the result of the Michael tandem addition / annulation reaction with cleavage of the constituents from the resin to produce the desired phenols 39. The synthesis is initiated using a polymer, obtained from Merrifield resin by attaching sodium to 3-hydroxypyridine to obtain higher-bearing resin 44, which is successfully quaternized with 1-bromopropan-2-one (or 2-bromo-1-phenylpropan-1- on; 2 bromo-1,2-diphenylethanone; 2-bromo-1-phenylbutan-1-one; 3-bromobutan-2-one) to obtain a polypyrimidine 43 salt. The reaction of polypyrimidine 43 with α, β-unsaturated ketones is carried out for 16 h after filtering the resin create a library 39.

References:

1. Those !, 32, 6937, (1993).

2. a. R. Αίρ £, Te!. Bey., 36, 7819, (1995);

B. K. ye \ uppbo \\ yk |. I. Hundred. Sket. 1, 105, (1999).

3. Ό. Ogesk !, He1y. Sket. Ac! A, 65, (1997).

4. Skepega, AO 95 / 16712,1995.

5. Sotroipbk 9 apb 21 ake k! Ab1e ίη 1kek sagbopu1! Ai! ke! egosusk Sket. 3. (1984).

6. Rkagta / 1e, 5435, (1999).

7. I. Do Not! Hisoscus Sket. 24, 589, (1987).

8. I. Not! Hisosus11s Sket. 23, 1821, (1986).

9. Sket. Bk !. 90, 121631y, (1979).

10. Mop! Acc Sket 107 587 (1976).

11. Those !, 48, 5473, (1992).

12. a. A. Mage / t / Pu I. Ogd. Sket., 63, 723, (1998); B. AO 9815532; from. 81b. Kyt / 2k. 87, (1991);

b. I. Not! His Hubus Sket. 24, 1141, (1987).

13. a. 8. Ko1odae), Te!. Be !!., 37, 5277, (1996); B. 8Up! Keky, 86, (1985).

14. a. Ό. Roteegk, Te! Hakebgop, 54, 4085, (1998); B. K. 1! O, I. Not! Hisosusks Sket., 29, 1037, (1992).

15. a. V. Natreg, Te!. Ley., 40, 4973, (1999); B. C. Sksh, I. Sot. Sket. 1, 73, (1999).

16. a. M. Oogbeeu, Te!. Be !!., 37, 4643, (1996); B. 8. The tablet, I. Sot. Sket. 1, 184, (1999).

17. T. Maediekp, He1y. Sket. Ac! A, 646, (1998).

18. I. Not! His Hubus Sket. 25, 959, (1988).

19. a. Those!. Be !!. 38, 211 (1997); B. I. Furniture Sket. 39, 4156, (1996); from. 8Up! Kek1k, 147, (1986).

20. Those!. Be !!., 6557, (1998).

21. 8bk! Yi! 1op o £ 4-ki1Pnu1 bepuyayu take OH \ νί11 1eab! O 4-rupt1bop. ί. Not! Hisoskus Sket. 22, 49, (1985).

22. Ka1gp1hku A., Those !. Le !!., 39, 8051, (1998).

16.2 6,6,5,6-cyclic framework of indolo [2,3-b] quinoline

The synthesis scheme of indolo [2,3-b] quinolines 1a, b is given below.

The key step in this synthesis is the decomposition of the corresponding triazoles 2a, b in polyphosphoric acid (PPA) at 110-160 ° C, which gives the desired compounds 1a, b ^1,2 . Isomers 2a and 2b can be separated during purification. The starting triazoles 2a, b can be obtained by heating the trisubstituted chloroquinolines 3 with the building blocks of benzotriazole 6a, b at 110-120 ° C in the presence of TEM ^1.3 . The building block of benzotriazole 6a, b is obtained from monosubstituted nitroanilines by reduction of the ΝΌ ₂ group (8pC1 ₂ or H ₂ / Pb) and the subsequent diazotization of the easily obtained diamines ^1,4 .

- 68 010258

Synthesis of indolo [2,3-b] quinoline

2-chloroquinolines 3 are obtained in three stages from substituted anilines, first anilides are synthesized either by reaction with previously activated (BTC, ΌΜΆΡ, collidine) β-keto acids or with a free acid at high temperature followed by intramolecular cyclization of compounds 5 under acidic conditions. In conclusion, the obtained quinoline is chlorinated with freshly distilled POC1 ₃ ⁵ to obtain compound 3. Another approach, namely, solid-phase synthesis of compounds 1a, b, can be carried out using disubstituted anilines with functional groups (СО ₂ Н, ΝΉ ₂ , ОН) attached to solid substrate.

The starting anilines can be loaded onto a suitable resin according to the type of functional group to be attached. If the functional group is CO ₂ H, the resin may be phenolic (see the quinoline chapter, changing the composition in accordance with the format of this description) and loading is carried out under esterification conditions (BTC, ΌΜΆΡ); if the functional group is OH, loading can be performed by the Mitsunobu reaction; and if the functional group is IN ₂ , the starting anilines can be charged under sulfonation onto a sulfonyl chloride resin or, al

- 69 010258 ternatively obtained by Kurtius rearrangement from the corresponding carbonyl derivatives.

References:

1. B1oogd. Meb. Siet., 7.2457, 1999.

2. Agsy Ryagsh 321463, 1988.

3. Those !. Yeah. 39 1827; 1998.

4. Ogd. 8 unit Co1. Yo1. 1 3 106

5. Ogd. 8 unit Co1. Yo1. 3 194

6. For other methods for producing indolo [2,3-b] quinoline, see

a. for acylbenzotriazole and acylisocyanate, I Ogd Set 65 8069 2000;

b. a combination of 3-bromoquinoline with 2-aminoboronate, 8nl 1067 1997;

c. by modifying the reaction of Gribe Ullmann, I. Meb Suite 37 3503 1994.

16.3 6,5,5,6-tetracyclic frameworks of isoindoloindoles and isoindoloindolones

In the present description, catalyzed P6 annelation ^{1 is} disclosed for the formation of the skeleton of isoindoloindole from easily obtained imines and internal aryl acetylenes. Imines and disubstituted acetylenes are subjected to a multi-step reaction in the presence of a palladium catalyst to form isoindoloindoles ² , which are obtained in good yield (see scheme below).

Isoindolindole

General scheme for the production of isoindoloindoles

By using different building blocks of either mono- or disubstituted iodanilines 7 and previously created di- or trisubstituted phenylacetylenes 5, a large library of isoindoles 1-4 can be created (see diagram below).

Such an annulation reaction contains two stages of synthesis without isolation of intermediate indoles 6. The stages of synthesis are as follows:

1. Imin 6 is formed in solution using drying reagents, such as TMOR, molecular sieves or Na ₂ 8O ₄ .

2. Acetylenes 5 are prepared using the Heck reaction between commercially available or pre-synthesized di- and monosubstituted iodobenzenes and monosubstituted acetylenes using standard P6 catalyst ^3-8 (see chart above). A ^9-12 modified Heck solid phase reaction may also be used. When using a solution, the reaction mixture can be used in the next step, as such, without reducing the catalyst, since it is required for the next step.

- 70 010258

3. Anneling of internal alkynes to isoindoloindoles using Pb (Oac) ₂ in the presence of the amine LiCl or Vi ₄ LiCl in DMF.

If one of the substituents is in the orthogonal position, the closure of the tetraz ring can occur in a regioselective manner, giving only tetrasubstituted isoindoloindoles 1,3. If the ortho assignment to 5 is not occupied, some substituents control the regioselectivity of the ring closure region by chelation of palladium in the intermediate σ-palladium, which is formed during the reaction. Other representatives of the two isomers can be isolated by chromatography. To create 11-hydroxyisoindoloindoles: TM8 protected by hydroxyalkine 11 can be used, creating after removing TM8 (η-Βι.ι ₄ ΝΡ) a sub-library of 11-hydroxyisoindoloindoles 12 (see diagram below).

can be used to get can

For the synthesis of 11-aminoisoindoloindoles, carboxyalkin 5

11-carboxyisoindoloindoles 1-4. The latter can be converted to the corresponding azodocarbonyl 14 (p-ViOCOS1, then ΝαΝ ₃ ) ¹³ ' ^-15 through an intermediate compound of aminoisoindoloindoles 13.

(see diagram below) that can be rearranged to nitrene to provide the desired sub-library 11-

A permanent polar functional group such as guanidine can be added. The most suitable position for this purpose is the para position on the phenyl ring obtained from imine 10 (see diagram below). Imine 10 carries a protected Vros amino group, the protection of which can be quickly removed after the formation of a ring with a suitable alkyne to produce indole 9. Amino isoindoloindole 9 can be reacted with bis-Thiourea 16 (NdC1 ₂ , TEM) to create, after subsequent removal protection (TFU / OSM), final library 8.

- 71 010258

16.3.1 Isoindoloindolones

The slightly modified framework of isoindoloindolone (see below) can be formed in two ways of synthesis:

K2

A schematic description is shown in the diagram below:

The approach presented above is divided into three large stages:

1. The formation of di- or trisubstituted indoles by the Heck reaction between acetylene and iodaniline.

2. Benzoylation of the indole ring with an orthioiodobenzoyl group. The addition of disubstituted orthioiodobenzoic acid BB to indole 18 can be carried out in two ways:

1. The usual addition of explosives to indole using OSS / OMAR ¹⁷ ;

2. The use of pre-synthesized acid chloride ^18,19 .

3. Cyclization using a catalyzed P6 reaction (Heck annihilation) ^20,21 . Attachment is very specific using the iodobenzoyl ring. If position 7 is not occupied, then it is possible to join indole in position 7 instead of position 2. This connection gives a new framework, which is a different library (see diagram below).

Indole 18 can be obtained by traceless solid-phase synthesis of indole using indole Ν-H as the resin attachment point ²² , which can be cleaved to obtain free indole 18. One of the most effective methods for the synthesis of indole in solution is Pb (O) the indirect reaction of 2-iodanilines with acetylenes in the presence of a base, as developed by Laroc. Monosubstituted 2-iodaniline is then loaded into a THP resin using an amine bond using PPT8 to give compound 20 (see diagram below). Replacement catalyst Pd (RRy _{3) 2} C1 ₂ and the use of soluble EFE base VOLUME found that it is helpful to promote the reaction to completion annulation to give 19. Cleavage of the resin with 10% TFA may then give the free indole 18. It was found that TM8-substituted acetylenes quickly reach completion at 80 ° C with almost complete regioselectivity.

Carboxylated 15 (B ₂ = CO ₂ H) can be converted to an analog of amine 16 corresponding to azodocarbonyl, which can be rearranged via an intermediate nitrene compound to provide the desired library of aminoisoindoloindolone.

Hydroxy and carboxyisoindolones 23 (X = O, CO ₂ ) can be formed using 8P synthesis (see diagram above), starting with loading the appropriate iodaniline onto resin ⁹ and annihilating TM8 with acetylenes.

Subsequent benzoylation and anneling of compound 27 followed by cleavage from the resin gives compound 25.

The second way of formation of isoindoloindolones is presented in the following scheme ²⁵ :

The key stage is the Wittig external molecular reaction. Substituted orthoalkylanilines and phthalic anhydride derivatives react to form arylphthalimides. They can be converted to phosphonium salts and can be combined into a system of isoindoloindolones.

References:

1. Lathosk B. I. At. Siet. 8os. 121, 3238, (1999).

2. Voeksi K. Ogd. Ley., 1551, (1999).

- 73 010258

3. Masbopa1b S. Siet. Soshtip. 2647, (1996).

4. Ata! Oge S. 1. Ogd. Siet. 60, 6829, (1995).

5. Ata! Oge S. 1. Ogd. Siet. 61, 8160, (1996).

6. Lauac! Ge O., Te! Gaybgop, 53, 7595, (1997).

7. Sa1 M. 8up1eys Sottip. 27, 1935, (1997).

8. Wah! Apaye T. 8upye !!. 207, (1992).

9. United States M. Those !. Be !!. 38, 7963, (1997).

10. Those !. Yeah. 38, 2307, (1997).

11. Those !. Yeah. 38, 2439, (1997).

12. Ata! Oge S. 1. Ogd. Siet. 61, 5169, (1996).

13. Katea1 V. Those !. Yeah. 35, 4947, (1994).

14. Skik K. Those !. Yeah. 36, 7193, (1995).

15. Ra1k 8. Those !. Yeah. 37,5303, (1996).

16. Aydaba V. Vuogdats & Mebkta 1 Sietkhyu, 2487, (1999).

17. Kgaik S. 8up! Ye! 1s Sottip. 23, 55, (1993).

18. Co / 1co \\ 8k1 A. Those !. Yeah. 32, 3317, (1991).

19. B1ask Ό. Te! Gayebgop 49,151, (1993).

20. 8yao N. Those !. Be !!. 39, 7235, (1998).

21. Oekagige E. Not! State 41, 1987, (1995).

22. 8th A. Te !. Yeah. 39, 8317, (1998).

23. Lagoske K. 1. Ogd. Siet. 60, 3270, (1995).

24. Lagoske K. 1. Ogd. Siet. 63, 7652, (1998).

25. 1. Not! Hisosusek Siet. 21, 623, (1984).

16.4 Monoatomic framework

The smallest framework used in such an implementation is a monatomic framework, namely, a framework of one carbon, of the general structure a:

but

The library contains several sub-libraries of ith (see below), which are represented by compounds with one constant functional group and independent substituent variants around carbon:

Ab NOT nz ^Ρ1 \ -Α2 ^but * ^with 1s X Κ1 K2 b FROM ϋ e

Secondary sub-libraries containing two or three constant polar functionalities (see below) may be somewhat limited due to the chemical instability of molecules carrying two or three attached to the same amine atoms or hydroxyl atoms (compound E):

141 ^{n0 bn} \ -Ή2. η νΆ ^{n 'm} CHN _g About 1 Η ₂ Ν he ABOUT L but he ί d to one one

However, the synthesis of α-amino acids k, α-hydroxyacids, and α-dicarboxylic acids 1 is known. For example, they are described by Koyey M. Uyyatk in 8puye818 о £ orysayu asyu α-atpo as1bq, Regdatop Rheck.

Some of the carbon-based compounds are the most commercially available. Those that are not commercially available can be synthesized, mainly in solution, using conventional methods.

The tertiary alcohols th ¹ can be synthesized using the well-known epoxidation of olefins 2, as a key step in the production of epoxides 1, which already have the necessary substituents ² (see diagram below).

- 74 010258

Groups of electron donors usually increase speed. The conditions are mild and the output is high. Subsequent reduction of epoxides is easily accomplished. The most common reagent is L1L1H ₄ , which reacts by inverting the configuration of compound 2 ³ . As expected from mechanism 8-2, cleavage usually occurs such that the desired tertiary alcohol b is formed. Product b serves as a starting material for tertiary amines c, which are obtained from product b by replacing the corresponding trifluoromethyl sulfonate with ammonium in dioxane. Solid phase preparation of tertiary alcohol L has recently been published ⁴ . Indeed, such a new cleavage strategy involves the addition of carbon nucleophiles to ester-linked resins 3.

• wh ABOUT H2MdH THF <Ether nsggdy cmI ⁺ background 3 b

Using this technique, only tertiary alcohols with two identical alkyl or phenyl substituents (B2) can be obtained, thus limiting the variety of products, but still suitable for quickly creating a secondary sub-library of tertiary alcohols.

α-hydroxy acids t can be obtained using a direct one-step procedure from the corresponding α-keto acids 4 (scheme below). α-Oxo acid 4 is commercially available, and its treatment with Grignard reagents (2 eq., THF, -40 ° C - CT) leads to the desired products.

Schiff base-activated glycine supported on soluble resin (PEC) 6 can simply be alkylated with a wide variety of electrophiles in the presence of a carbonate base (C8 ₂ CO3) in acetonitrile ⁵ , providing non-stereospecific amino acid esters.

Similarly, Schiff base activated amino acid tert-butyl esters 8 can be alkylated to α-C disubstituted analogues 7 (the scheme is shown below) using alkyl bromides and LOL as the base (LOL, THF, -40 ° C).

Schiff 8 bases can be prepared by transimination of the commercially available tert-butyl ester of amino acids 9 with benzophenolimine. Finally, the alkylated product may be completely unprotected with TFA / OSM, giving the desired secondary sub library.

It should be noted that all products created in this chapter are enantion-selective and

- 75 010258 require the separation of enantiomers on a chiral column. The use of racemic mixtures may also be considered.

References:

1. Thea Nebgoy, 2855, (1976).

2. The visa. Oh. Ne., 956, (1985).

3. I. Ogd. SN., 52.14, (1981).

4. 8. СНаибгазекНаг, I. Sot. SN., 2,246, (2000).

5. a. B. 8aiuadia1, Those !. Ley., 39, 821, (1998) .; B. B. 8aiuadia1, I. Sot. SN., 2.134, (2000).

16.5 6.7 bicyclic framework of benzodiazepines

Benzodiazepines are therapeutic agents and anticonvulsants. For example, such 1,4-benzodiazepines are targets of several solid-phase synthesis strategies.

The synthesis of 1,4-benzodiazepines is based on the closure of the seven-membered ring through lactamation in high yield.

The slightly modified solid-phase approach, which is based on ring closure through the imine group, is shown in the diagram below:

According to such a strategy, aldehyde resin 1 ^{3 is} coupled to β-amino alcohol 2 via reductive alkylation (FIG. 1). β-aminoalcohol (2) can be obtained in two alternative ways (see chart below):

(1) The combination of Ν-methoxyhydroxamate (8) with Grignard reagents (H ₂ MdVg) to obtain the corresponding ketones followed by reduction using NaBΗ ₄ (MeOH, room temperature, several hours) to obtain a Boc-protected amino alcohol derivative (9). Removal of the protecting groups gives compound 2.

(2) Recovery of Ν-metoksigidroksamata (8) 1L1N ₄ to the aldehyde derivative, followed by coupling with Grignard reagents (B MdVg ₂₎ to form the Boc-protected aminoalcohol (9). Removal of the protecting groups gives compound 2.

The combination of the aldehyde resin (1) and the hydrochloride salt of an amino alcohol (2) is carried out through reductive alkylation using NaΒΗ (Oac) 3, 1% AcOH, DMF to obtain β-amino alcohols 3 mobilized by the resin 3. In order to avoid racemization, it is desirable to obtain equilibrium between the aldehyde 1 bound to the resin and β-amino alcohols 2 until a reducing agent is added to the reaction mixture.

The combination of secondary amine 3 and Boc-protected disubstituted anthranilic acids 4 leads to an intermediate resin-bonded compound 5. Oxidation of the hydroxy group gives compound 6. Oxidation on a solid support can be carried out using the Ru8O ₃ ⁹ complex in DMSO at room temperature or using an alternative procedure using ΝΜΟ ¹⁰ (Ν-methylmorpholine Ν-oxide) with TPAP (tetra-n-propylammoniumperrutinate) catalyst in DMF at room temperature. Compound 6 is deprotected (TFA / OSM) and the free amine is subjected to intramolecular cyclization under acidic conditions to obtain the desired benzodiazepine 7.

The introduction of an amine or hydroxyl at position 3 of 1,4-benzodiazepines can lead to decomposition of the substance. At position 2, the OH group can be isomerized to form a ketone, while the ΝΗ ₂ group can form tautomers with the imine group.

The synthesis pathway for producing a benzodiazepine having a substituent ΝΗ ₂ at position 2 is presented in two ways below:

(1) The thioamino ester (10) is charged onto the aldehyde resin 1 by reductive alkylation (No. 1BH (OLe) ₃ , 1% AcOH in DMF) to obtain the intermediate 11 bound to the resin (FIG. 3). Secondary amines (11) bind to disubstituted anthranilic acids (12) (EOS, ΝΜΡ) to form amide 13, which can be subjected to intramolecular cyclization using ligated p-methoxyacetonylide (14) ¹ to obtain thiobenzodiazepine 15. Cyclic resin-bound intermediate 15 subjected to methylation (Me1), followed by oxidation to form the preferred leaving group (namely methyl sulfoxide) for nucleophilic substitution. Such substitution reactions can be performed with acid labile dimethoxybenzylamine (16) under standard conditions (DMF, ΌΙΕΆ), providing, after acid cleavage, the desired sub-library of 2-aminobenzodiazepines 17.

(2) An alternative synthesis of 2-aminobenzodiazepine is as follows: benzodiazepin-2,5-dione (20) is formed by combining substituted anthranilic acid with an amino acid followed by ring closure, which is reacted with the Lawesson reagent to form intermediate 2-thiobenzodiazepin-5-one (21). Amine 22 is prepared by reacting benzodiazepintion 21 with ammonium.

Alternative synthesis of 2-aminobenzodiazepine

- 77 010258

The synthesis of 3-hydroxy-a-amino acids, the building block used to produce 2 carboxybenzodiazepine, is shown in the diagram below. The commercially available chiral tert-butyl ether of Ethos-Serine 27 is subjected to Swern oxidation ((C0C1) ₂ , DMSO) to obtain aldehyde 27. Aldehyde 27 is subjected to the Grignard K. | MDX reaction to form Ethos-protected amino alcohols, which after removal of Ethos (piperidine, MeOH) lead to the desired building blocks 28. In the case where both K. ₁ and _II are both carboxylic groups, the starting material is di-tert-butyl fumarate 23, which upon epoxidation (tCPBA, Να№0 ₃ , ESM) gives epoxide 24 followed by exposure to ammonia in methanol for I am getting compound 25.

СО ₂ (Vi tSRVA, ΝβΗΟΟ,. DSM

CO _{2 1} Vi

ΝΗ ₃ in MeOH p ^ CO ^ Vi ------ СО ₂ 1Vi

H ₂ M

CO _{2 1} Vi

CO ₂ (Vi

UN

РтосНЦ ^ СО ^ Ви

Corneal Oxidation

1. (1 equiv.) K.MdVg, THF

KtosNM ^ CO _g ) B _and ^g · Piperidine, MeOH

Κ1 ^ ΟΗ

Н ₂ Н ' ^хЧ СО ₂ ®и

Preparation of L-hydroxy amino acid

The synthesis of benzopyridodiazepine 33 ^11,12 is shown in the diagram below.

2-chloro-3-aminopyridines 29 ^{12 are} attached to the building block of the disubstituted azidobenzoyl chloride 30. The reduction of azide 31 8pC1 ₂ gives the intermediate 2-chloroxosolidine 32, which, when treated with acid, regroups into the desired tricyclic pyridine-based framework 33.

The synthesis of an oxyanalogue to framework 33, namely, 10H-dibenzo [b, G] [1,4] oxazepin-11-one, is shown in the diagram below. The disubstituted o-aminophenol binding unit 35 is attached to the resin on an acid-sensitive methoxybenzaldehyde (AMEBA) (34) by oxidative amination to form a resin 36.

Resin 36 is further modified with substituted 2-fluoro-5-nitrobenzoic acid 37 using the H0A1 / E1C strategy to obtain an immobilized substrate 38, which is ready to assemble nitro-10H-dibenzo [b, D] [1,4] oxazepine-11- analogues she 39 (key cyclization step ₍₈ 8- Ar) is performed using 5% ΌΒυ in DMF ^23,24,25 between fluorine and oxygen phenol). The reduction of the nitro group in the resulting resin can be carried out using a 1.5M solution of 8pC1 ₂ H ₂ O in DMF, and subsequent cleavage (TFA / OCM) from the resin creates a 2-amino library of compound 39.

- 78 010258

Synthesis of Dibenzooxazepinone

References:

1. 1. Ogd. SEet, 62, 1240, 1997,

2., 1SS. 2, 513, 2000,

3.8up1eys Sot., 21,167,1991,

4. 1. Ogd. SEet, 60, 5742, 1995,

5. Those !. Ley, 39, 7227, 1998,

6. 1. Ogd. SEet, 63, 8021.1998,

7. Those !. Ley, 37, 8081, 1996,

8. 1. Ogd. SEet, 60, 5744, 1995,

9. 1. At. Sows. 8os, 116, 2661, 1994,

10. 1. Ogd. SEet, 61, 8765.1996,

11. 1.Not1.Set., 23, 695, 1986,

12. 1. Ogd. SEet, 62, 6102, 1997,

13. Those !, 55, 2827, 1999,

14. Those !, 55, 8295, 1999,

15. The1. Ley, 40, 5827, 1999.

16.6 6,6,6-tricyclic pyrazinoquinosalinone framework

"1 K4

The pyrazino [2,1-b] quinosaline-3,6-dione system can be considered as a linked peptidomimetic, which is present in some families of natural products. Some of these compounds exhibit very interesting biological activity (1. ApyOysk 46, 380, 1996, Appi Keu B1ocet 62 385.1993).

One of the known syntheses of such frameworks can now be grouped as follows:

a: Transformation of 4-substituted 2,5-piperazinediones into the corresponding imino ethers followed by cyclocondensation with anthranilic acid or methylanthranilate ^1-5

Condensation of anthranilic acid imido ether with pyrazinoquinosalinone

B: Acylation of the 4-substituted 2,5-piperazinedione with o-azidobenzoyl chloride followed by the reaction of Staudinger with phosphine to obtain the corresponding γ-phosphazine and cyclization of Wittig with azavnutrimolecular replacement of the latter intermediate. ^6.7

- 79 010258

Pyrazinoquinazalinone via Ν-o-azidobenzoyldiketopiparasin

In the sequence of the modified reaction, Ν-o-azidobenzoyl diketopiparasine is formed through an open-chain tripeptide, where the anthranilic acid element is the Ν-terminal element bearing the azido group as a masked amino function ⁸ . Cyclization creates a chinozalinone ring.

c: Double cyclization of an open chain tripeptide through an intermediate 4-imino4-H-3,1-benzoxazine, which is carried out by cyclohydration of a suitable oacylanthranilamide in the presence of iodotriphenylphosphine.

Such a method is described in solution ^9-13 , as well as solid phase ¹⁴ , which makes it a good tool for parallel matrix synthesis and, therefore, suitable for the purpose of the present invention.

Tripeptide 6 is obtained by direct combination of amino acid esters (AA-OK) 3 with anthranilic acid mediated by EBS. The condensation of compound 4 with Ethos-amino acid chloride 5 under the two-phase Scott-Bauman reaction (CH ₂ Cl ₂ , aq. NoCO ₃ ) gives tripeptide 6. Amino acid chloride 5 is obtained by pre-activation of the corresponding Etos-AAOH BTC (triphosgene) and collidine in THF, BSM or dioxane ¹⁵ . Such conditions give AA chlorides without racemization.

The transformation of the linear tripeptide into oxazine is carried out under Vip conditions (PP13 / 12 / large excess of tertiary amine). Deprotection followed by rearrangement to quinazoline is carried out by treatment with 20% piperidine in methylene chloride. Cyclization in chinosaline is susceptible to spatial noise and in the case of K3, K4 = volume groups, cyclization requires stringent conditions (BMAP reflux ί.Ή ₃ ί.'Ν). In some examples, some epimerization occurs (5%).

The use of synthesis 8 in the solution described above in combinatorial solid-state synthesis begins with loading the Wang resin with a suitable amino acid (AA), which gives compound 7. Most AA preloaded onto the Wang resin are commercially available. Compound 7 is deprotected (piperidine in DMF) and only suitable anthranilic acid is coupled (EBS) to the resulting compound 8 (the scheme is shown below).

- 80 010258

The next step is the acylation of aniline 7 Ethos-AA-C1 to obtain a linear tripeptide 9. The next step is a key cyclization with dehydration of the linear tripeptide 9 to 10. To be sure of complete conversion, use 10 equivalents of P11 ₃ P. The final reaction is piperidine-mediated removal of the protective Ethos groups and rearrangement of oxazine 10 to amidine carbamide 11. After washing, the resin is refluxed in acetonitrile to initiate cleavage with cyclization of compound 11 to create a gel library pyrazinoquinazolines 1. The yield and purity of the crude compounds are declared, respectively, above ¹⁴ . Final products 1 can be obtained in some cases as a mixture of cis: trans diastereoisomers (usually a ratio of 5-8: 1). A higher degree of solid phase epimerization is usually the result of cleavage with cyclization, and an NT purifier can separate products. The above synthesis perfectly illustrates the advantages of this synthesis path. The first two stages include the addition of peptides - a reaction for which 8PP8 was developed and which proceeds with practically quantitative yield for many amino acids. Dehydration of the linear peptide 9 requires a large excess of P11 ₃ , P. iodine and TEM reagents, which are quickly removed by simple filtration on a solid phase. The ester functionalization group that underwent cyclization in the last step is selected as the position for the solid phase addition resulting from self-cleavage from the resin.

The synthesis of the framework of pyrazinoquinazoline requires 3 building blocks, two amino acids 3,5 and disubstituted anthranilic acid 2.

Amino acids and Ethos-amino acids are commercially available.

To introduce heterofunctional groups (ΝΗ ₂ , OH) into the pyrazine ring (K3, K4), it is necessary to synthesize protected α-hydroxy-AA 14 and α-amino-AA 12. AA 12 is well known from literature ¹⁶ and the synthesis is illustrated in the diagram below:

Toluene, reverse heating

Yeti ΝΗ

Toluene, heating with reflux r-To-OH NSMVi.

r-T "-ON

Η & -ίΒυ

Synthesis of protected cx-amino-a-OH amino acids

Another AA 14 can be obtained in a similar way through condensation between glyoxylic acid and осtocNΗ ₂ in the presence of tert-ViOH while boiling in toluene to obtain the desired 14.

- 81 010258

From the third building block, 3,5-dimethylanthranilic acid is commercially available, another substituted anthranilic acid must be synthesized with desired properties. 3-methyl-5-phenylanthranilic acid 15 can be obtained by bromination of commercially available 3-methylanthranilic acid 16 ¹⁷ . Then follows the reaction of Suzuki ¹⁸ .

3,5-diphenylanthranilic acid 17 can be obtained from the corresponding dibromanthranilic acid 18 (commercial) using a P6-catalyzed cross-linking reaction with an excess of phenylboronic acid ¹⁹ (Aldrich).

vg _a , meon ⁱⁿ yy ^{so, n}

4% ------- Τ ' ¹ · ¹ W

Aldrich Aldrich

RNV (OH) a (3 equiv.) P <1 (RRK _E) ) ₄ 10% M YOUN, vod. ₃ CO ₃ ^{P | 1}

Reflux, h 17

Getting diphenylanthranilic acid

Substituted anthranilic acid can also be obtained from the corresponding substituted aniline 19 using a modified Sandmeier procedure. The reaction of aniline with chloral or hydroxylamine gives isonitrosoacetanilide followed by cyclization in sulfuric acid to give isatin 20. Then oxidation of H ₂ O ₂ gives anthranilic acid ²⁰ 21 (see diagram below).

Anthranilic acid 22, substituted at position 3 by the OH group, can be obtained by the following sequence of reactions shown in the scheme below, using 4-substituted anilines (Εΐ, Pr, Me Aldrich) as starting materials. First, aniline is brominated to compound (23), followed by selective monomethoxylation in the presence of Cu1. Thus obtained 2bromo-6-methoxy-4-alkylaniline 24 carbonylated using P6 complex as catalyst (Co, P6 (RRy _{3) 2} C1 ₂₎ (~ 25) and the last step is deprotected by hydrolysis in concentrated hydrobromic acid ²¹ .

- 82 010258

4-alkylaniline 19a can also serve as a starting material for the production of dialkylanthranilic acid 26 and 5-alkyl-3-phenylanthranilic acid 27, as shown in the diagram below.

3-alkyl-5-carboxylanthranilic acid 27 can be obtained starting from o-alkylaniline 19b, which is converted to isatin 20a (1. chloral, ΝΗ ₂ ΟΗ, 2. H ₂ 8O ₄ ), followed by bromination and oxidation to obtain 5 β-bromanthranilate 28. Substitution of bromine with cyanide and hydrolysis of compound (29) gives 3-alkyl-5 carboxylanthranilic acid ²² 27.

References:

1. TePayigop Akut 9 3025, 1998.

2. TePayigop Akut 11, 3515, 2000.

3. TePayigop 55 14185, 1999.

4. TePayigop 54, 969, 1998.

5. TePayigop Akut 113515, 2000.

6. .1AC8121 11953, 1999.

7. TePayigop 57 3301, 2001.

8. GOS 65 1743, 2000.

9. GOS 63 2432, 1998.

10. Te1gayeygop Lei 40, 5429, 1999.

11. Ogd. Yeah. 2, 3103, 2000.

12. GOS 65.1022, 2000.

13. 1. Ogd. Siet., 63, 2432, 1998.

- 83 010258

14. 1. Honeycomb. Sib., 2.186, 2000.

15. a. 1. Rerybe Kez., 53, 507.1999. B. Those! Le!., 34 3861, 1993.

16. Proc. Νίώ. Asab. 8οΐ. I8A, 93, 2031, 1996.

17. Those! Yeah. 41, 21083, 2000.

18. 1. At. Eat. 8os. I2, 2707, 2000.

19.8Un! Le515 14101995.

20. a. 8Up! Sottip. 29, 3627, 1999

b. 1. Ogd. SJ 59, 6823, 1994,

c. 1. Meb. Eat. 34.1896, 1991,

b. 1. 1pb1ap. Eat. 8os. 66, 39, 1989,

e. Those! Bie !! 29, 3709, 1988,

1. 1. Meb. Svet 30.1166, 1987.

21. 1. Meb. Eat. 25,267, 1990.

22. Those! Gayebgop, 50, 2543, 1994.

16.7 Frame 5-membered pyrrole ring

This chapter describes the complete synthesis of tetra-substituted pyrroles. The proposed synthesis methods are solid phase (8P8), as well as in solution.

A sub-library having a carboxyl group at position 2 is prepared in solution. The synthesis begins with nitrosation of β-ketoesters to produce oximes 3, which by reductive condensation of 1,3-diketones lead to ethyl carboxyketopyrroles 5 ¹ (Scheme below). Pyrroles 5 are subjected to reduction of the carbonyl group to methylene ¹ , followed by hydrolysis of ethyl carboxylate to create a sub library a. Kurtius rearrangement can convert carboxyl to amine, resulting in the conversion of sub-library a to sub-library L in the most convenient way (in the case of CJC2, mixtures of two isomers are obtained that can be separated).

To create a sub-library a and b, two building blocks of β-ketoesters, 1,3 diketones, which are the most commercially available, are required.

Library compounds c can be obtained by the synthesis method shown in the scheme below.

- 84 010258

In contrast to the developer's method, the proposed approach involves solid-phase synthesis (8P8). Namely: the condensation of 1,2-diketones 7 with pre-attached monoester of Vosiminoacetoacetic acid 6, as follows:

Reaction ^{2,3 is} carried out under basic conditions using NaOe or Co1Bi. Iminodiacetoacetic acid 6 can be easily obtained from the tert-Vi ester О1у by reductive amination of glyoxylic acid using sodium cyanoborohydride as a reducing reagent and the subsequent introduction of the Boc-protecting group in a multi-gram amount of ⁴ .

Sub-library e can be created using the method shown in the diagram below. Position 3 in the resulting products has a fixed substituent hydroxy group. Again, 8P8 was carried out using previously obtained building blocks, as described above.

The method begins with the creation of five building blocks of the acylated acid of Meldrum (12) ^5.6 in solution by the reaction of acid chlorides 10 with Meldrum acid 11 to obtain in the presence of pyridine the corresponding compound 12 almost quantitatively ^7.8 .

Thus, heating compound 12 (5 equiv.) With a hydroxyl resin (a resin that generates carboxylic acid, for example, oxime resin ⁹ ) in THF, while refluxing for several hours ^6, gives β-ketoesters 13 associated with the resin at concomitant release of CO ₂ and acetone, which helps to bring the reaction to completion. The reaction can be easily controlled by ET-1B on resin (KBg granules). The functionalization of the α-carbon group of compound 13 was carried out with excess alkylation reagent, avoiding O-alkylation as well as double alkylation.

Thus, haloalkanes (36 eq.) In the presence of 1M TBAE ⁸ in THF (26 eq., 3 h) readily convert compound 13 to compound 14 at RT (Fig. 4). It is usually important to eliminate traces of water, which may reduce yield. An additional excess of pre-synthesized aminoketones 15 ^10.11 (Fig. 5) (20 eq., 3 h, CT) for resin-bound β-ketoesters 14 in a THF / trimethylorthoformate mixture (1/1) gives Schiff bases 16. Cyclization of Schiff bases 16 under alkaline conditions with the concomitant release of product 17 into solution and subsequent reduction of the ketone (B3 = Me, E1) (NaBH ₄ BE ₃ OE1 ₂ ) ^! create a sub-library e.

- 85 010258

The reaction can also be carried out in solution using α-substituted β-ketoesters. What follows is the same sequence of reactions.

It should be noted that β-hydroxypyroles can exist to some extent in the form of their ketotautomer ¹⁴ . The required building blocks are β-ketoesters that are commercially available, or α-substituted β-ketoesters.

The building block of α-aminoketone can be obtained from the corresponding amino acid, as shown in the diagram below.

P1-MdH, THF

I 9 9 ^m ® -20 ° С 'room I 9р, / Ц.'У' ³ ± Е ^{, е} 2! ^iox . ^{n e} s.n'-oo

1> 1920

Synthesis of Aminoketones from C1u Boc-Hydroxamates

Ν-protected glycine reacts with Ν-O-dimethylhydroxylamine to give hydroxamate 18. It is observed that the reaction of glycine hydroxamate with Grignard reagent (E £ MdVg, MeMdVg) is able to give ketone 19 without adding an excess. Removing protection from compound 19 gives the building blocks of aminoketones.

In the case of K3 = OH, glycinate reacts with substituted β-ketoesters.

More than sixteen products can be obtained by the method shown in the diagram below. A key step in creating the £ sub-library is the addition of Michael 21 aminoketones to ΌΤΆΌ (21) ¹² . The obtained amino olefins 23 are cyclized under acidic conditions to create a sub-library £.

H2

heating

The synthesis of 2-carboxy-3-aminopyrroles 28 is well known ^12,13 (see diagram above). It is carried out by the formation of enamine from compound 26 and subsequent intramolecular cyclization of 27 in basic conditions (ΝηΘΕί) to obtain compound 28. β-ketonitriles 25 can be obtained by easier alkylation of 25 ¹⁵ or acylation of the corresponding nitrile ¹⁶ .

References:

1) I. Rat III, I. Ogd. Siet., 3857, (1976).

2) M. Eyebtap, I. Ogd. Siet., 859, (1965).

3) K. ΟιιιιιόιΙι. App Siet., 639.102, (1961).

4) S. Wook, I. Ogd. Siet., 5687, (1992).

5) b. T1e1he. Vogd. & Meb Siet. Yeah. 1303, (1997).

6) b. T1e1 / e. 8ΥΝΙ.ΕΊΊ '. 661, (1996).

7) Υ. O1ka \\ a. I. Ogd. Siet., 2087, (1978).

8) b. Aeh. 8 ¥ \ 'KETT. 1156, (1998).

9) Thye Sotta! Opa1 1pbeh, p. fifteen.

10) 8. Νηΐιιη. Those!. Ley., 3815, (1981).

11) Eig. I. Ogd. Siet. 2809, 2000.

12) N. Aagb. Those!. Ley., 25, 527, (1969).

13) Mi-111 Yt, I. Ogd. Siet., 3826, (1979).

14) Ah! I. Siet. 20, 935, 1967.

15) I. Ogd. Siet. 55 429 1990.

16) a. Vi11. Siet. 8os. 1rp. 62 3851 1989; B. Siet. Ryagt. Vi11. 46 69 1998; from. I. Furniture Siet. 34 1741 1991

16.8 Thiophenes and related scaffolds

The chemistry of 2-aminothiophenes and related scaffolds has attracted particular attention over recent years due to its use in pharmaceuticals, agriculture, pesticides and dyes.

The chemistry of 2-aminothiophenes is readily available due to the synthesis method developed by Geowold ^1ab , which proposed the easiest and most promising synthesis pathway leading to 2-aminothiophosphenes A with substituents that select electrons, such as cyano, carbetoxy, etc. in positions 3 and the groups of alkyl, aryl, cycloalkyl and heteroaryl in positions 4 and 5.

Gewald's reaction

The simplest version of the Gewald reaction contains a single-capacitance technique, namely, the use of condensation of aldehydes, ketones or 1,3-dicarbonyl compounds with activated nitriles and sulfur in the presence of an amine at room temperature. Ethanol, DMF, dioxane are preferred solvents, and amines like diethylamine, morpholine or triethylamine ^{1-7 are used} . This method offers a significant improvement over other methods by replacing α-mercaptoaldehyde or α-mercaptoketone with simpler starting materials. It is necessary to use 0.5-1 molar equivalents of amine based on the amount of nitrile to obtain a high yield. In another version of the synthesis, a two-step procedure is preferred. a, Unsaturated nitrile is first obtained by Knevenagel-Kope condensation and then treated with sulfur and amine. Such a two-stage version of the Gewald reaction gives higher yields. Alkyl aryl ketones do not give thiophenes in a single-capacitance modification, but give acceptable yields in two-step method ² (see diagram below).

Gewald's two-stage reaction

The use of tert-butyl cyanoacetate instead of ethyl ester makes it possible to obtain the free acid of 3-carboxy-2-aminothiophenes by conventional TFA / ESM hydrolysis ⁸ .

An amino acid obtained in the same way as a protected acid can be used as building blocks for further transformation into more complex scaffolds, as illustrated below.

16.8.1 5,5-bicyclic frameworks

reverse heating

Κ, ΟΟ, .ΜβΟΗ / Η, Ο or oktgadnl

IN 1

The thienopyrrole framework B ⁹ (scheme above) is formed by the reaction of aminocarboxylate A with bromoacetate (K2CO3) to obtain the diester intermediate compound 1, which, after acetylation (compound 2) (30% AcCl in AcOH), is subjected to Dieckmann condensation (ЕУ№1. Е1ОН. Е1ОН). ) to obtain 3-hydroxy-2-carboxyteno [2,3-b] pyrrole B1. The synthesis of amino analog B2 should begin with 2-amino-3-cyanothiophene A1. Acetylation followed by alkylation with abromoacetate (K ₂ CO ₃ in acetone or NaΗ in DMF) under similar reaction conditions leads to ring closure, forming 3-aminocarboxythienopyrrole B2. To increase the nucleophilicity of the amine, acetylation of the amine at position 2 and the UN is required.

- 88 010258

16.8.2 5,6-bicyclic frameworks

The thienopyridine framework C is created by a modified Fredlander reaction, namely, the reaction of thiophene A, A1 and 5 with complex L-ketoesters, 1,3-diketones under basic conditions for

Another 5,6-bicyclic ring system - thienopyrimidine Ό, is obtained by the reaction of thiophene A, A1 with chloroformamidine hydrochloride 4, 11.

16.8.3 5,8,5-, 5,8,6-tricyclic and 5,5,8,6-, 5,5,8,5-tetracyclic frames

Frames E, E, O, and H can be formed from thiophenes shown in Scheme 6. Such compounds are obtained by forming the eight-membered ring of dilactam.

- 89 010258

The formation of an eight-membered ring involves several stages:

1. Activation of the β-amino acid using 8OC1 ₂ ^12a or POC1 ₃ ^12b (in these cases, the amine must be protected by Boc) or using OCC ^12c and methyl chloroformate ¹²⁴ .

2. The combination of activated acid and other Ν-protected complex (3-amino-tert-butyl ether ¹³ .

3. Deprotection of tert-butyl ester and Ν-Boc amine using TFA in PSM.

4. The combination with the help of RuVor or any other analogue, if K. ' is a benzyl group, at this stage it can be removed by hydrogenation.

16.8.4 5,7-bicyclic framework

The synthesis of I, 1 analogues of benzodiazepine scaffolds is illustrated by the following scheme. In two approaches, a chiral amino acid is introduced into the synthesis, increasing the diversity around α-carbon. Thienodiazepine I is obtained from 2-amino-3-acylthiophenes 5, which react with previously synthesized Boc-amino acid chloride (amino acid, BTC, collidine, THF or ESM). Deprotection of compound 8 (4n. HC1) with concomitant ring closure results in 2oxothienodiazepine I. Thiophenodiazepine ί can be prepared starting from 2-amino-3-carboxythiophenes A, which, after preliminary activation with thienoxosinedione (BTC, collidine, THF or ESM) react with aminoketone to obtain 9, ring closure gives 5-oxothienodiazepine I ¹⁴ .

- 90 010258

Synthesis of Thienodiazepine K

The synthesis of thienodiazepine K is shown in the diagram above. 2-amino-3-acylthiophene 5 is first acetylated with a suitable nucleophilic substitution of α-haloacetyl chloride ΝαΙ, then with ammonia to obtain aminoamide ¹⁵ 11. The ring is then closed in thienodiazepine K under acidic conditions. Another alternative is the reaction of thiophene 5 with a phthalide protected by aminoacyl chloride, deprotection of hydrazine (11) and ring closure to obtain thienodiazepine K ¹⁶ .

The synthesis of thienozepine L is based on the combination of succinic anhydride or the acid chloride monoester with thiophene 5 (see diagram below). The resulting amide 12 is subjected to intramolecular condensation (No. H) to obtain the target compounds ¹⁷ .

A framework M having a skeleton of thienodiazepinone can be obtained as shown in the diagram below. The защищ-protected aminocarboxythiophene A is first preactivated (BTC, collidine, ESM) and reacted with α-aminoacetonitriles 14 to obtain amide 13. Then, under basic conditions (No. OMe), the reaction is carried out under intramolecular cyclization of the substituted intermediate 2-aminothieno -1,4-diazepin-5-one 15 ¹⁸ . In the next step, 2 aminothieno-1,4-diazepin-5-one 15 is heated with acetylhydrazine, which leads to thienotriazolodiazepinone M.

- 91 010258

16.8.5 5,6,5,6-tetracyclic and 5,6,5-tricyclic frameworks

Thiophene substituted at position 3 with benzimidazole, namely benzimidazoloaminothiophene 16, can serve as a building block for the synthesis of thieno (2 ', 3', 4,5) pyrimidino (1,6) benzimidazole N, N1. The starting material, 2-cyanomethylbenzimidazole 16, was prepared from substituted phenylenediamine 17 and malonitrile ¹⁹ . Nitrile 18 is subjected to a Gewald reaction using elemental sulfur powder and ketones ²⁰ or cyanoacetamide ²¹ in dry DMF containing a catalytic amount of TEM under reflux to form thiophene 16 (see diagram below).

Condensation of 16 with aldehydes and ketones gives N and N1, respectively ^21.22

- 92 010258

Using the same approach, dihydroimidazoylacetonitrile 20 ²³ (see chart above) and thienoimidazoylacetonitrile 21 (see chart below) can be obtained from the corresponding diamines (ethylenediamine and thiophen-2,3-diamine ²⁴ ) and malononitrile. The resulting nitriles react with ketones under the conditions of Gewald’s condensation, forming O, O1 and P, P1.

16.8.6 5-6-5-6-tetracyclic framework

4H-thieno [2 ', 3': 4,5] pyrimido [2,1-b] benzothiazoles or benzenes O can be obtained from aminothiophene A, as illustrated in the scheme above ²⁵ . 2-amino-3-carboxythiophene is condensed at

- 93 010258 high temperature with chlorobenzimidazole ²⁶ . Chlorobenzimidazole 23 leads to the corresponding thienopyrimidinazoles O.

16.8.7 5-6-5-tricyclic framework

Thiatriase-8-indacenone K (see chart below) can be prepared according to the procedures described in the literature. In this synthesis, aminothiophene A is cyclized in boiling acetic acid with preformed methylthioimidazoles 24 to obtain the desired system K.

References:

1. a. Sows. Veg. 99 94 1966, b. P11agta / 1a 51833, 1996.

2. 1. Do not! Hisosus1. Sows. 36333, 1999.

3. Vi11. Sows. 8os 1r. 64 3768.

4. Vuogd. May. Sows. Yeah. 7 1629 1997.

5. 1. Sows. Here. Vu! Espo1 47p39 1990.

6. Mopa! 127,297,1996.

7. 1py1ap 1. SEet. 1209 1971.

8. a. 1. May. Sows. 41 1729 1998,

b. Those! Bie !! 40 5471 1999,

c. 1. Negosus 1. Sows. 32 1537 1995.

9. Vi11. 8os. Sat. Eh. 1786 1975.

10. a. Sows. Reag. Vi11. 47 993 1999,

B. Reokreogik 8i1Eig 8Shopop apy ge1a! Her e1epep! K 155 215 1999.

11. a. 1. Not! Hisosus1. Sows. 9 775 1972, b. 1. May SEet 16 191 1973.

12. a. He! Sosusek 27 105 1988;

b. Ogd. Rger. gosey. 1p! 29,711 1997;

c. 1. Sows. 8os. Regkt Tgapk. 1 1649 1982;

th. Eok1 Akai№iki88K41 1989.

13. a. Ace! Asai. δα Nypd 107 171 1981,

B. 1yp1ap 1. SEet. 8es! In 16B 393 1978.

14. 1. Do not! Hisosus1. Sows. 16 793 1979.

15. Yeig. 1. May. Sows. 31,683 1996.

16. a. 1. May. Sows. 16,214,173;

B. So11es! Sekhsk SEet. Sottip. 49 621 1984.

17. a. 1. May. SEet., 18, 192, 1975;

b. 1. Not! Hisosus1. SEet., 36, 477, 1999;

c. 1. Not! Hisosus1. SEet., 33, 271, 1996.

18. a. Exit App. SEet., 328, 1979;

B. 1. Η. e! egosus 1. SEet., 29, 1477, 1992.

19. 1. At. Sows. 8os. 65 1072 1943.

20. Reokreogik 8i1Eig 8Shopopy gi1a! Ei e1etep! K, 105, 51, 1995.

21. Mopa! Keeee SEet., 127, 955, 1996.

22. Reokreogik 8i1Eig 8ShopApy Ge1a! Ei eteep! 106, 193, 1995.

23. Ace! And sows. 8 sleep. 50 432 1996.

24. 1. Sows. Kekeages. 296 1985.

25. Yeig. 1. May. SEet., 29, 569, 1994.

26. a. Ike! 1. Sows. 35 775 1982,

b. Eagtaso 44 227 1989,

c. Eig. 1. May. Sows. 24,623 1989.

27. 1. May. Sows. 30 1166 1987.

28. 1. Not! Hisosus1. SEet., 38, 743, 2001.

It should be noted that the above methods for measuring targets and finding drugs

- 94 010258 substances may vary in various ways, including changing the order of the stages, which stages are performed online, which stages are performed offline. Additionally, various parallel and / or serial configurations can be used to implement the present invention, optionally using a variety of software tools and / or various combinations of software and hardware. Additionally, not all of the distinguishing features shown above in a particular embodiment are necessary in each such illustrative embodiment of the present invention. In particular, it is contemplated that a combination of the above distinguishing features is also included within the scope of certain embodiments of the present invention. Also included in the scope of the present invention is a computer-readable medium on which software is written to execute part or all of an illustrative embodiment of the present invention. You should also take into account that many variants of the present invention are described only as methods or only as devices. The scope of the present invention also covers hardware and / or software adapted and / or designed and / or programmed to implement process type options of the present invention. Additionally, the scope of the present invention includes methods of applying, constructing, performing calibration, and / or servicing the devices described herein. The headings where they appear are provided to facilitate viewing and should not be construed as limiting the content of the sections to the meaning of the specified headings. The use of the terms in the following claims contains, contains, includes, includes, has or their root words means including without limitation.

It will be apparent to those skilled in the art that the present invention is not limited to this description. On the contrary, the scope of the present invention is limited only by the following claims.

Claims (59)

CLAIM 1. A method of obtaining information about at least one chemically active region of at least one target molecule, comprising providing a set of compounds comprising a plurality of substantially rigid chemical meters, each of which is comprised of a rigid framework and at least one a fragment attached to it by a single covalent bond, which is free in terms of rotation; each of said plurality of meters provides at least one substantially rigid triangular geometric substructure of the tie points, so that at least 50% of the virtual space is covered by essentially rigid triangular geometric substructures of said meters, and each point of said space is covered by at least at least 5 different meters from the specified set; said virtual space is defined using a set of at least six chemical binding sites, including acidic, basic, hydrophobic functions, a hydrogen bond donor, a hydrogen bond acceptor, an aromatic ring, and the range of possible distances between chemical binding sites includes a range from 2 to 12 A , the specified set and the specified interval define the space of possible triangular configurations, each configuration includes a triplet of distances within the specified interval, forming t eugolnik and triplet binding types for the vertices of the triangle; a portion of said space is defined as being covered if at least one substantially rigid triangular substructure exists capable of chemically bonding with the triangular configuration of the binding points selected in said portion; the interaction of at least one specified target with the specified set of meters; measuring the interaction of these meters with at least one of these targets; analysis of these research results to obtain information about the specified at least one chemically active region. 2. The method according to claim 1, in which the space includes a section in the range from 4 to 8 A, the specified section is covered by at least 50%. 3. The method according to claim 1, comprising selecting at least one of said ranges, and said plurality of meters is based on at least one said target. 4. The method according to claim 1, in which the specified coverage covers at least 50% of the points of the specified space, which includes the base. 5. The method according to claim 1, in which the analysis contains a characterization of the specified chemically active region of the target. 6. The method according to claim 5, wherein the characterization comprises identifying a configuration that matches the configuration of the associated meter. 7. The method of claim 5, wherein the identification comprises a configuration identification that does not match the configuration of the associated meter. 8. The method according to claim 7, in which the identification includes identification using a statistical analysis of these research results. 9. The method of claim 8, wherein the identification comprises identification by clustering. 10. The method according to claim 5, in which the identification contains the assumption that each meter indicates a single configuration. 11. The method of claim 5, wherein the identification assumes that at least some meters indicate a plurality of configurations. 12. The method according to claim 5, in which the identification contains a classification of meters by chemical groups at the vertices of these configurations. 13. The method according to claim 5, comprising reconstructing a spatial map of at least a portion of said chemically active region according to the results of at least two experiments, said portion comprising at least four chemical binding regions. 14. The method according to item 13, in which the specified part includes at least six chemical binding regions. 15. The method of claim 12, comprising reconstructing the spatial map of at least a portion of said chemically active region in at least two of these identified configurations, said portion comprising at least four chemical binding regions. 16. The method of claim 15, wherein said portion comprises at least six chemical binding regions. 17. The method of claim 13, wherein the reconstruction comprises a test reconstruction of a plurality of spatial maps from said configurations; assessment of these maps and the selection of a spatial map based on its assessment. 18. The method of claim 13, wherein the reconstruction comprises a test reconstruction of a plurality of spatial maps from said configurations; clustering these maps according to general structures and selecting a spatial map based on the relative property of the cluster to which it belongs. 19. The method according to p, in which the specified compared property includes size. 20. The method according to item 13, in which the specified spatial map includes enough binding points to ensure the binding of the drug substance in the form of a small molecule having a chemical profile corresponding to the binding points. 21. The method according to claim 20, in which the specified spatial map includes at least 6 points of binding. 22. The method according to item 21, in which the specified spatial map includes at least 8 points of binding. 23. The method according to claim 1, wherein said plurality of meters comprises a set of meters with at least 10,000 meters. 24. The method according to claim 1, wherein said plurality of meters comprises a set of meters with at least 50,000 meters. 25. The method according to claim 1, in which the specified coverage covers at least 50% of the points of the specified space, which includes acid. 26. The method according to claim 1, in which the specified space includes a section in the range from 4 to 12 A, said section is covered by at least 50%. 27. The method according to claim 1, in which the specified space includes a plot in the range from 8 to 12 A, said plot is covered by at least 50%. 28. The method according to claim 1, in which at least 0.5% of these meters is associated with at least one specified target. 29. The method according to claim 1, in which at least 1% of these meters is associated with the specified target. 30. The method according to claim 1, in which at least 3% of these meters is associated with the specified target. 31. The method according to claim 1, in which at least 0.1% of these meters is associated with at least one target. 32. The method according to claim 1, in which at least 50% of these meters is determined by adding groups to a set of less than 100 frames. 33. The method according to claim 1, in which at least 50% of these meters is determined by adding groups to a set of less than 50 frames. - 96 010258 34. The method according to claim 1, in which at least the specified set of meters uses less than 15 different chemical groups to determine the chemical behavior of these meters. 35. The method according to claim 1, in which at least the specified set of meters uses less than 10 different chemical groups to determine the chemical behavior of these meters. 36. The method according to claim 1, wherein said study is a functional study. 37. The method according to claim 1, wherein said study is a binding study. 38. The method according to claim 1, wherein said study is a cell study. 39. The method according to claim 1, in which the specified research is a continuous study. 40. The method according to clause 36, in which the specified functional study is performed in the presence of a natural substrate of the specified target. 41. The method according to claim 1, wherein said target contains a protein comprising a biochemically active region adapted to adhere to the substrate. 42. The method according to paragraph 41, wherein said chemically active region comprises a region comprising said biochemically active region. 43. The method according to paragraph 41, wherein said chemically active region comprises a control region of said protein. 44. The method according to claim 1, in which the analysis contains an analysis of the successful binding of at least 60 meters. 45. The method according to claim 1, in which the analysis contains an analysis of the successful binding of at least 10 meters. 46. The method according to claim 1, in which the analysis contains an analysis of the successful binding of at least 100 meters. 47. The method of claim 5, wherein the identification comprises identifying at least 40 different configurations. 48. The method according to claim 5, in which the identification comprises the identification of at least 10 different configurations. 49. The method of claim 5, wherein the identification comprises identifying at least 100 different configurations. 50. The method according to clause 16, including comparing the specified card with a database of leaders and selecting a leader from the specified database for further use, sensitive to similarity or lack of similarity between the specified leader and the specified card. 51. The method according to clause 16, including comparing the specified card with a database of leaders and the deviation of the leader from the specified database from further use, sensitive to the similarities between the specified leader and the specified card. 52. The method according to clause 16, including the construction of a leader having similarities with the specified map. 53. The method according to paragraph 52, in which the design includes construction using these meters or frames used to determine these meters. 54. The method according to claim 5, comprising comparing said configurations with a leader database and selecting a leader from said database for further use that is sensitive to matching said configurations with said leader. 55. The method according to claim 5, comprising constructing a leader based on these configurations. 56. The method according to claim 5, comprising selecting at least one of these meters as a leader for finding a drug substance. 57. The method according to claim 1, comprising comparing the binding of the meters with a similar geometry binding to obtain spatial inconsistencies data and analyzing said spatial inconsistencies data to provide geometric information about the specified target. 58. The method according to claim 1, comprising creating a set of drug leaders for the specified target based on the specified information. 59. The method according to § 58, including the removal of known drug leaders for the specified target from the specified set.